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系統識別號 U0026-0108201118575600
論文名稱(中文) 賴胺酸與酪胺酸嵌段共聚物及其經雙糖修飾之嵌段共聚物:分子自組裝與藥物傳輸
論文名稱(英文) Lysine-block-Tyrosine Block Copolypeptides and Corresponding Disaccharides Modified Block Copolypeptides: Supramolecular Assembly and Drug Delivery
校院名稱 成功大學
系所名稱(中) 化學工程學系碩博士班
系所名稱(英) Department of Chemical Engineering
學年度 99
學期 2
出版年 100
研究生(中文) 楊曜聲
學號 n36981456
學位類別 碩士
語文別 中文
口試日期 2011-07-13
論文頁數 94頁
口試委員 口試委員-張鑑祥
口試委員-林睿哲
口試委員-吳文中
指導教授-詹正雄
關鍵字(中) 聚胺基酸
分子自組裝
奈米沉降法
液胞
關鍵字(英) polypeptide
self-assembled
nanoprecipitation
vesicle
學科別分類
中文摘要 近十年來,具有自組裝能力的材料被研究學者開發成載體粒子,比起微脂粒,雙親性高分子開發的載體具有較多優勢,如:仿生功能、誘發生化機制、藥物控制釋放能力及穩定性高的高分子載體粒子。1本文研究將胺基酸合成為嵌段共聚物,一端為聚賴胺酸(PLL)一端為聚酪胺酸(PLT)。PLL-b-PLT被設計成雙親性材料,由奈米沉降法在不同的溶液環境下可自組裝成超分子,如在DIW與pH12水溶液及磷酸鹽緩衝溶液等環境。經圓二色光譜儀鑑定PLL-b-PLT在DIW與pH 12下的二級結構,分別會從無規高分子纏繞變成β-摺疊、從α-螺旋變成無規高分子纏繞。隨分子量的改變,動態光散射儀器檢驗出PLL-b-PLT組裝的粒子粒徑分佈範圍在100nm 到400nm之間;使用穿隧式電子顯微鏡與靜態光散射儀可驗證PLL-b-PLT粒子的構型,發現不同分子量的PLL-b-PLT能分別組裝成微胞及液胞。具有組裝能力的PLL-b-PLT可用來包覆各種範例藥物:焦油腦、5-氟尿嘧啶、紫杉醇、阿黴素等,而從TEM觀察到包覆藥物的PLL-b-PLT粒子會因為和藥物的π鍵堆積力或氫鍵作用力使載體粒子在變形成長蟲狀的奈米粒子。PLL-b-PLT載體材料經過修飾乳醣酸後具有標靶人體肝臟細胞的能力,本研究使用化學修飾法將乳酸糖修飾在PLL-b-PLT上,讓組裝成的高分子載體提升了生物相容性及外加標靶治療的能力。2, 3
英文摘要 In decades, polymersomes which consist of self-assembled materials have received much attention from the sciencetist as nanoscale encapsulants. Compared with liposomes, developing novel amphiphilc copolymer for nanoparticles formation has much potential on material properties, such as stimuli responsiveness , induced biofunctionality, drug control release, and stability. On this study, we used polypeptides, polylysine and polytyrosine, as both segments of di-block copolymer. PLL-b-PLT designed as a zwitterionic diblock copolymer can form nanoparticle by nanoprecipitation in different solution condition, neutral or basic aqueous. Circular dichroism measurement shows that PLL segments adopt random coil conformation and change to β-sheet, PLT segments change from α-helical structure to random coil when pH value goes from 7 to 12. DLS shows that PLL-b-PLT has broad size distribution, 100nm~400nm, which was also evidenced by TEM figure. Different molecular weight of PLL-b-PLT demonstrated the shape of nanoparticle, micelle or vesicle, by TEM and SLS. After morphological characterization, the interaction between polymeric carrier and model drugs, pyrene, 5-fu, TAX and DOX, was investigated, also the encapsulation efficiency and loading capacity of PLL-b-PLT. TEM figure shows that when PLL-b-PLT complex with model drug, the spherical polymersomes change the conformation to worm-like nanoparticle. In the research of drug delivery system, Lactobionic acid can target the certain organ in human so we modified the target ligand on the side chain of PLL. Thus, the polymeric carrier formed by PLL-b-PLT can enhance the properties: biocompatible and targeting ability.
論文目次 摘要 I
Abstract II
誌謝 IV
目錄 VI
表目錄 IX
圖目錄 X
第一章、緒論 1
1.1 奈米藥物傳輸系統:目的與動機 1
1.2 奈米藥物傳輸系統:技術與材料 1
1.3 奈米藥物傳輸系統:市場需求性 2
1.4 奈米藥物傳輸系統:發展癌症藥物載體 3
第二章、文獻回顧 4
2-1 奈米載體的材料 4
2-1-1微脂粒介紹 4
2-2 高分子藥物載體 6
2-2-1 高分子藥物載體起源 6
2-2-2 高分子藥物載體組裝與結構分析 7
2-3聚胺基酸藥物載體 10
2-3-1 聚胺基酸 10
2-3-2嵌段共聚胺基酸的設計與生化功能性 12
2-4高分子藥物載體包覆與釋放 16
2-4-1包藥製程法 16
2-4-2控制釋放 17
第三章、實驗 19
3-1實驗藥品 19
3-2 實驗方法 21
3-2-1 PLL-b-PLT合成 21
3-2-2分析樣品的製備 28
3-2-3檢測藥物包覆率與釋放效果 32
3-3 實驗儀器 36
3-3-1 GPC 36
3-3-2 NMR 38
3-3-3 UV/Vis 39
3-3-4 CD 40
3-3-5 PL 41
3-3-6 DLS 42
3-3-7 TEM 43
3-3-8 SEM 44
3-3-9 SLS 45
第四章、結果與討論 46
4-1嵌段共聚胺基酸的樣品定義 47
4-1-1 以凝膠滲透層析儀(GPC) 分析PZLL分子 47
4-1-2 分析嵌段共聚胺基酸的分子組成 52
4-1-3 使用NMR搭配GPC分析PZLL-b-PBLT的組成 54
4-2 PLL-b-PLT的自組裝條件與結構型態探討 57
4-2-1臨界聚集濃度(Critical aggregation concentration)的測量 57
4-2-2 以NMR鑑定PLL-b-PLT在不同環境下的構型 60
4-2-3 以CD及UV/Vis檢驗PLL-b-PLT的 二級結構 62
4-3 分析並定性PLL-b-PLT奈米載體 65
4-3-1製備奈米粒子:奈米沈降法(Nanoprecipitation) 65
4-3-2製備奈米粒子: 穩定母液稀釋法 70
4-4:鑑定裝載不同藥物的PLL-b-PLT奈米粒子 76
4-4-1裝載藥物的奈米粒子: 穩定母液稀釋法 76
4-4-2 PLL-b-PLT以奈米沉降法包覆藥物與鑑定包覆量 81
4-5 使用PLL-b-PLT及其雙糖衍生物包覆DOX 85
4-5-1 用NMR與SLS鑑定K66Y30-g-La 85
4-5-2使用超音波震盪包覆Doxorubicin 86
第五章、結論 88
第六章、文獻回顧 90
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系統識別號 U0026-0108201211245800
論文名稱(中文) 聚賴胺酸與酪胺酸無規性共聚胺酸:自組裝與其奈米載體及包覆體應用
論文名稱(英文) Poly(L-lysine)-r-Poly(L-tyrosine) Random Copolypeptides: Self-Assembly and Their Applications as Nanocarriers and Nanoencapsulants
校院名稱 成功大學
系所名稱(中) 化學工程學系碩博士班
系所名稱(英) Department of Chemical Engineering
學年度 100
學期 2
出版年 101
研究生(中文) 呂承勳
學號 n36991184
學位類別 碩士
語文別 中文
口試日期 2012-07-06
論文頁數 68頁
口試委員 指導教授-詹正雄
口試委員-陳東煌
口試委員-侯聖樹
口試委員-王勝仕
關鍵字(中) 無規性共聚物
聚胺基酸
複合奈米粒子
奈米金
自組裝
液胞
微胞
關鍵字(英) polypeptide
composite nanoparticles
nanogold
self-assemble
vesicle
micelle
學科別分類
中文摘要 合成不同親疏水比例及不同分子量的聚賴胺酸與酪胺酸無規性共聚胺酸 (poly(L-lysine)-r-poly(L-tyrosine), (KY)),在水溶液下,共聚胺酸會自組裝形成結構。藉由穿透式電子顯微鏡(Transmission Electron Microscopy)檢測,發現在不同分子量下,分別會形成液胞及微胞。透過動態光散射分析儀(Dynamic Light Scattering)量測,聚胺酸形成的奈米粒子粒徑在40到120奈米之間,與穿透式電子顯微鏡所觀測的粒徑相符。經圓二色光譜儀(Circular Dichroism)檢測,不同親疏水比例聚胺酸具有不同的二級結構。經照射紫外線的聚胺酸奈米粒子,其酪胺酸會二聚化(dimerization)形成交聯,使奈米粒子更加穩定,並且不影響粒徑大小。除此之外,利用酪胺酸與四氯金酸的反應,也會使聚胺酸奈米粒子被交聯。交聯過程中,tyrosinate ion 將電子傳給金屬離子後,形成tyrosyl radical,最後tyrosyl radical二聚化形成雙酪胺酸。雙酪胺酸可利用UV/Vis及螢光光譜儀進行檢測:UV/Vis檢測下,雙酪胺酸會在320到350奈米產生吸收;而由螢光光譜儀檢測,在325奈米的激發光下,雙酪胺酸在410到430奈米具有螢光。肌紅蛋白(myoglobin)可以被包覆在聚胺酸液胞內。利用加入四氯金酸,可以製備出包覆肌紅蛋白的奈米金/聚胺酸複合液胞。由穿透式電子顯微鏡及小角度X光繞射儀(Small Angle X-ray Scattering)確定,內核(core)及外層(shell)分別是由肌紅蛋白及奈米金/聚胺酸構成。DLS顯示,包覆肌紅蛋白的奈米金/聚胺酸複合奈米液胞的粒徑約78奈米。聚胺酸奈米粒子及奈米金/聚胺酸複合奈米粒子,在觸媒、生物標靶(biomarkers)、藥物載體(drug carriers)、仿生性包覆體(biomimetic encapsulants)及功能性奈米反應器(functional nanobioreactors)等方面深具潛力。
英文摘要 poly(L-lysine)-r-poly(L-tyrosine) (KY) amphiphilic random copolypeptides with different hydrophilic-to-hydrophobic ratios and molecular weights were synthesized. Transmission electron microscopy (TEM) analysis revealed that KY copolypeptides self-assembled to form vesicles or micelles in solution, depending on KY molecular weight. Dynamic light scattering (DLS) confirmed that the hydrodynamic diameters of KY nanoparticles were ranged between 40 and 120 nm, consistent with those determined by TEM characterization. Circular dichroism measurements showed that the secondary structure adopted by KY was determined by the Lysine to Tyrosine ratio. KY nanoparticles can be stabilized via dimerization of tyrosine residues under UV irradiation. In addition, KY nanoparticles also can be cross-linked at basic pH by the reduction process between tyrosine and gold precursor. In this process, the tyrosinate ion donated an electron to the metal ion through the formation of a tyrosyl radical, which is eventually dimerized to form dityrosine during the reaction. The relative composition of gold in the peptide/gold shell can be tuned by varying the tyrosine to lysine ratio in the copolypeptide. DLS analysis showed that dimerization of tyrosine did not cause the change of KY nanoparticle size. The cross-link of KY nanoparticles through the dimerization of tyrosine was confirmed by UV/Vis and fluorescence analysis. UV/Vis analysis confirmed that the presence of dityrosine as evidenced by the absorbance band between 320 and 350 nm. Fluorescence analysis revealed that the emission band of dityrosine between 410 and 430 nm upon excitation at 325 nm. The model protein, myoglobin, was encapsulated in KY vesicles and the protein-loaded gold/polypeptide composite vesicles was prepared after adding gold precursor. TEM and Small-angle X-ray scattering(SAXS) analysis confirmed that the core and shell comprised of myoglobin and gold/polypeptide analysis revealed the size of the protein-loaded gold/polypeptide composite vesicles was about 78 nm. With the unique feature of the copolypeptides, these as-prepared polypeptide and polypeptide/metal composite nanoparticles will be useful as catalysts, biomarkers, drug carriers, biomimetic encapsulants, and functional nanobioreactors.
論文目次 第一章 緒論 1
第二章 文獻回顧 3
2.1 胺基酸聚合 3
2.1.1 NCAs合成 3
2.1.2 NCAs開環聚合 4
2.2 聚胺酸雙親性高分子 5
2.2.1 聚胺基酸高分子類型 5
2.2.2 應答機制 9
2.2.3 應用 13
2.3 交聯反應 14
第三章 實驗 16
3.1 實驗藥品 16
3.2 實驗儀器 17
3.3 PLL-r-PLY合成 23
3.3.1 乾燥溶劑 23
3.3.2 合成起始劑 23
3.3.3 製備 NCA 24
3.3.4 聚合反應 25
3.3.5 移除保護基 26
3.4 PLL-r-PLY奈米粒子製備 27
3.4.1 DLS樣本製備 27
3.4.2 TEM樣品製備 28
3.4.3 SAXs樣品製備 28
3.5 奈米金/聚胺酸複合奈米粒子製備 28
3.5.1 奈米金/聚胺酸複合奈米粒子 28
3.5.2 包覆肌紅蛋白的奈米金/聚胺酸複合奈米粒子 28
第四章 結果與討論 30
4.1 無規共聚胺基酸樣品分析鑑定 30
4.1.1 使用GPC鑑定PLL-r-PLY分子量 30
4.1.2 NMR分析PLL-r-PLY組成 31
4.2 PLL-r-PLY的臨界聚集濃度量測 34
4.3 PLL-r-PLY自組裝粒徑及結構檢測 35
4.3.1 酪胺酸交聯成雙酪胺酸的方法 35
4.3.2 交聯後奈米粒子的粒徑及構型 37
4.3.3 離子強度對自組裝粒徑影響 46
4.4 利用CD進行PLL-r-PLY進行二級結構的檢測 48
4.5 聚胺酸與四氯金酸形成有機/無機複合粒子 52
4.5.1 奈米金/聚胺酸複合奈米粒子 53
4.5.2 包覆肌紅蛋白的奈米金/聚胺酸複合奈米液胞 57
第五章 結論 64
參考文獻 65
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系統識別號 U0026-0206201321402100
論文名稱(中文) 重症病患適宜的營養支持
論文名稱(英文) Optimal Nutrition Support in Patients with Critical Illness
校院名稱 成功大學
系所名稱(中) 生命科學系碩博士班
系所名稱(英) Department of Life Sciences
學年度 101
學期 2
出版年 102
研究生(中文) 黃秀華
學號 l58961048
學位類別 博士
語文別 英文
口試日期 2013-05-11
論文頁數 115頁
口試委員 指導教授-張素瓊
口試委員-林少琳
召集委員-陳志毅
口試委員-張子明
口試委員-羅慧珍
關鍵字(中) 重症病患
疾病嚴重度
灌食途徑
早期灌食
周邊靜脈營養
支鏈胺基酸
關鍵字(英) critical illness
severity of illness
feeding route
early feeding
hypocaloric peripheral parenteral nutrition
branched-chain amino acids
學科別分類
中文摘要 重症病患主要以腸道及靜脈兩途徑給予營養支持。許多研究探討不同的灌食途徑對於病患營養狀況的影響,以及重症病患在進入加護病房48小時內給予早期餵食(early feeding),是否可以降低腸胃道耐受不良的機率,改善臨床結果;然而上述研究,很少有以疾病嚴重度為基準來探討腸胃道灌食及早期餵食對重症病患臨床結果的影響。支鏈胺基酸可提升免疫功能,被部分醫師用於腸胃道癌症術後恢復期的營養支持,但是否真正有利病情亦存有爭議。故本研究以重症病患為對象,探討在疾病嚴重度的影響下,不同的灌食途徑及灌食開始時間的早晚,與病患的營養狀況及臨床結果之相關性;同時也以大腸直腸癌的病患為對象,探討富含支鏈胺基酸的周邊靜脈營養對於術後臨床結果的影響。整體研究之目的為提昇重症病患的醫療品質,改善其營養狀況,以避免併發症的發生,節省醫療資源。
實驗ㄧ是探討疾病嚴重度是否影響灌食途徑對重症病患臨床結果的功效並決定適宜重症病人的灌食途徑。101位內科加護病房的重症病患隨機分配到鼻胃(n = 51)或鼻-十二指腸(n = 50)灌食途徑,病情的嚴重度以『急性生理和慢性健康評估Ⅱ』(APACHE II)為評估標準,分為“較不嚴重(APACHE II<20)”和“極重病(APACHE II>=20)”兩組。在21天的研究期間,收集每日熱量和蛋白質攝取量、灌食併發症、加護病房住院天數、死亡率、氮平衡、血清白蛋白和血清前白蛋白等數據。研究結果顯示,在較不嚴重組的病患中,鼻胃灌食及鼻-十二指腸灌食兩組在每日熱量和蛋白質攝取量、灌食併發症、加護病房住院天數及氮平衡都無顯著性差異。在極重病組的病患中,鼻胃灌食比鼻-十二指腸灌食顯示較低的熱量和蛋白質攝取量,較多的灌食併發症,較長的加護病房住院天數,及較差的氮平衡。故為讓營養支持更為適當,且考慮到醫療資源的有效運用,建議病情較不嚴重的患者使用鼻胃灌食,極重病組的患者使用鼻腸灌食。
實驗二是釐清疾病嚴重度與灌食開始時間之相關性。108位內科加護病房的重症病患進入加護病房48小時內開始灌食者屬於“早期灌食”(n = 40),超過 48小時則屬“晚期灌食”(n = 68)。病情的嚴重度以APACHE II 20分為區分標準,分為“較不嚴重 (APACHE II<20)”和“極重病(APACHE II>=20)”兩組,於研究21天期間,收集病患每日熱量及蛋白質攝取量、血清白蛋白及前白蛋白、24小時尿素氮、灌食併發症、住加護病房天數、住院天數、呼吸器相關性肺炎發生率、死亡率等數據。研究結果顯示,極重病組的患者,早期灌食比晚期顯示較佳白蛋白及前白蛋白、較差氮平衡、較多灌食併發症、較長加護病房住院天數。早期灌食可以改善極重病者之營養狀況,而灌食併發症及住加護病房天數雖劣於晚期灌食,但灌食併發症可被控制而不影響營養攝取量,而最終之住院天數及死亡率無顯著差異。故早期灌食相較於晚期對於極重病者仍是較佳之營養治療方式。
實驗三是探討富含支鏈胺基酸的周邊靜脈營養對於癌症術後病患恢復之臨床效益。這個觀察研究是將61位入院接受大腸直腸癌手術的病患,依照術後使用靜脈營養的支鏈胺基酸含量多寡,分為控制組“葡萄糖液”,低劑量組“葡萄糖液加低量支鏈胺基酸及脂肪滴劑”,高劑量組“葡萄糖液加高量支鏈胺基酸及脂肪滴劑”等三組,收集手術前ㄧ天至術後第7天的各項臨床數據。這些病患術前營養狀況正常,整個7天觀察期間,控制組的身體質量指數比低劑量及高劑量兩組,有明顯減少的現象;且控制組比高劑量組有較少的尿素氮流失及較低的氮平衡下降現象,而其他的臨床結果如免疫力、生化檢驗值、靜脈炎、住院天數及死亡率,控制組與其他兩組比較,均無顯著差異。因此,不建議術前無營養不良的大腸直腸癌患者,術後恢復期間給予富含支鏈胺基酸的周邊靜脈營養支持。
綜合以上結果,疾病嚴重度影響灌食途徑的有效性,並與灌食開始時間的早晚有密切相關;且富含支鏈胺基酸的周邊靜脈營養,無助於術前無營養不良的大腸直腸癌患者的術後臨床結果,此資訊可助醫療團隊改善重症病患的營養支持。
英文摘要 Patients with critical illness require either enteral or parenteral nutrition support. Many studies have investigated what the effects of using different feeding routes are on nutritional outcomes as well as whether the effect of early feeding can reduce the incidence of gastrointestinal intolerance and improve clinical outcomes in critically ill patients. However, effects of feeding on clinical and nutritional outcomes in patients with differences in illness severity have not been fully examined. Branched-chain amino acids are essential for lymphocyte responsiveness and support immune cell functions. Some physicians prescribe short-term peripheral parenteral nutrition with enriched branched-chain amino acids as nutritional support during the postoperative recovery in patients with gastrointestinal cancer. Whether this intervention provides better clinical benefit than routine management is still under debate. This study investigated the possible association between severity of illness and efficacy of enteral feeding route and the effect of commencement of enteral feeding on nutritional and clinical outcomes in patients with critical illnesses in a medical intensive unit and the effects of peripheral parenteral nutrition with high dosage of branched-chain amino acids on clinical outcomes in postoperative colorectal patients. The overall purpose of the study is to enhance the quality of medical care, improve nutritional status, avoid the occurrence of complications, and save medical resources in critically ill patients.

Experiment I-Different feeding routes
Experiment I was conducted to test whether severity of illness influenced the efficacy of enteral feeding route on clinical outcomes in patients with critical illnesses and determine the optimal enteral feeding route for different levels of severity.
One hundred and one patients in medical intensive care unit were randomly assigned to receive feeding through one of two routes, nasogastric (n = 51) or nasoduodenal (n = 50). These critically ill patients were dichotomized into “less severe” and “more severe” subgroups using a Acute Physiology and Chronic Health Evaluation (APACHE) II score of 20 as a cutoff. Daily energy and protein intake, feeding complications (gastric retention, vomiting, diarrhea, gastrointestinal bleeding), length of intensive care unit stay, hospital mortality, nitrogen balance, albumin, and prealbumin were collected over 21 days. In the less severely ill patients, there was no difference between the nasogastric and nasoduodenal groups in daily energy and protein intake, feeding complications, length of intensive care unit stay, and nitrogen balance. In the more severely ill patients, the nasogastric group had lower energy and protein intake, more tube feeding complications, longer intensive care unit stay, and poorer nitrogen balance than the nasoduodenal group.
These results suggest that gastric feeding route be used for less severely ill patients and postpyloric feeding route for more severely ill patients to optimize nutritional support and conserve medical resources.

Experiment II-timing of enteral feeding initiation
Experiment II was conducted to investigate the possible association between illness severity and commencement of enteral nutrition.
One hundred and eight medical intensive care unit patients receiving enteral feeding within 48 h of intensive care unit admission were assigned to an early feeding group (n = 40) and those receiving eternal feeding beyond 48 h of admission were assigned to a late feeding group (n = 68). Patients were further dichotomized into a less severe group (APACHE II <20) and more severe group (APACHE II >=20). Daily energy and protein intake, feeding complications (gastric retention, vomiting, diarrhea, gastrointestinal bleeding), length of intensive care unit stay, length of hospital stay, ventilator-associated pneumonia, hospital mortality, serum albumin, serum prealbumin, nitrogen balance, and 24-h urinary urea nitrogen were collected over 21 days. There were no differences in measured outcomes between early and late feedings for less severely ill patients. In the more severely ill patients, however, the early feeding group were found to have improved serum albumin and prealbumin but worsened nitrogen balance, more feeding complications, and prolonged intensive care unit stays than their late feeding counterparts.
These results demonstrated a relationship between illness severity and enteral feeding commencement time and the influence of this relationship on clinical outcomes. In more severely ill patients, early feeding was associated with improved nutritional outcome and late feeding with reduced feeding complications and shorter intensive care unit stays. The feeding complications of more severely ill patients started early on feeding can be handled without significantly affecting nutritional intake, and thus early feeding may be a more beneficial nutritional intervention than late feeding in patients with more severe illnesses.

Experiment III-Hypocaloric peripheral parenteral nutrition enriched with BCAA
Experiment III was conducted to examine whether the infusion of hypocaloric peripheral parenteral nutrition with high dose branched-chain amino acids provides better clinical benefits than routine fluid management in postoperative gastrointestinal cancer patients.
In this retrospective observational study, 61 colorectal cancer patients receiving postoperative hypocaloric peripheral parenteral nutrition were assigned to one of three feeding groups based on what nutrients they received: control group received dextrose infusion only, the low dose group received dextrose plus branched-chain amino acids and fat infusion and high dose group received dextrose plus high dose branched-chain amino acids and fat infusion. Outcome data were collected on the day before and seven days after surgery. Patients were non-malnourished. Over the observation period, the dextrose only control group had a significantly higher reduction in body mass index than the lower dose and the higher dose branched-chain amino acids groups. Compared to the high dose branched-chain amino acids group, the control group also had a lower urinary nitrogen loss and less reduction in nitrogen balance. There were no differences between the control and low dose/high dose groups in immunity, biochemical measures, phlebitis, hospital stay and hospital mortality. We found no clinical advantage to the postoperative administration of hypocaloric peripheral parenteral nutrition with branched-chain-enriched amino acids to non-malnourished colorectal cancer patients.
In conclusion, these three experiments showed that severity of illness influenced the effect of different enteral feeding routes and feeding commencement on nutritional and clinical outcomes. Postoperative hypocaloric peripheral parenteral nutrition with branched-chain-enriched amino acids provides no obvious benefits in non-malnourished colorectal cancer patients. These findings can be used by interdisciplinary health care teams to improve nutrition interventions for patients with critical illnesses.
論文目次 CONTENTS

Chinese Abstract I
English Abstract IV
Acknowledgements VIII
Contents IX
List of Tables XII
List of Figures XIII
Abbreviations XIV
Chapter 1 Introduction 1
Chapter 2 Literature Review 5
Nutrition support in critical illness 5
Energy and protein supply 5
Severity of illness 6
Enteral versus Parenteral nutrition 7
Enteral nutrition 7
Feeding complications 8
Parenteral nutrition 9
Supplemental parenteral nutrition 10
Feeding tube sites 10
Aspiration pneumonia 11
Energy target 11
Early versus Late feeding 12
Postoperative hypocaloric peripheral parenteral nutrition 13
Branched-chain amino acids 13
Hypocaloric versus Hypercaloric Parenteral Nutrition 14
Chapter 3 Experiments: Part I to III
I. Severity of illness influences the efficacy of enteral feeding route on clinical outcomes in critically ill patients
Abstract 16
Introduction 18
Materials and Methods 19
Results 24
Discussion 27
II. Association between illness severity and timing of initial enteral feeding in critically ill patients: A retrospective observational study
Abstract 37
Introduction 39
Materials and Methods 40
Results 43
Discussion 47
III. Hypocaloric peripheral parenteral nutrition with branched-chain-enriched amino acids provides no clinical advantage in well-nourished postoperative colorectal cancer patients
Abstract 56
Introduction 57
Materials and Methods 58
Results 62
Discussion 65
Chapter 4 Summary and Conclusions 74
Chapter 5 References 76
Appendixes 95
Publication List 98


List of Tables
Table 1-1. Comparison of demographic and clinical characteristics between nasogastric (NG) and nasoduodenal (ND) tube feeding groups and break down, by severity of illness 32
Table 1-2. Differences of measured outcomes between nasogastric (NG) and nasoduodenal (ND) feeding groups and break down, by severity of illness 33
Table 1-3. Effects of enteral feeding routes (nasogastric vs nasoduodenal) on measured outcomes after adjusting for gender, age and severity of illness in Acute Physiology and Chronic Health Evaluation II (APACHE II) 20 and APACHE II 20 group 34
Table 1-4. Incidence of tube feeding complications between nasogastric (NG) and nasoduodenal (ND) feeding groups in Acute Physiology and Chronic Health Evaluation II (APACHE II)  20 patients 35
Table 2-1. Demographic and clinical characteristics of the patients categorized by timing of feeding initiation and break down by illness severity 51
Table 2-2. Differences in measured outcomes between early and late feeding groups and break down by illness severity 52
Table 2-3. Differences in feeding complications between early and latefeeding groups categorized by illness severity 53
Table 2-4. Effects of the timing of enteral feeding initiation (early feeding) on different clinical and nutritional outcomes 54
Table 3-1. Composition of the PPN formula per group 70
Table 3-2. Comparison of patients’ characteristics between the control and experimental groups 71
Table 3-3. Differences in measured outcomes between the control and experimental groups 72
Table 3-4. Effects of HPPN with branched-chain enriched amino acids (high dose group) on different measured outcomes after adjusting BMI and NB on pre-operation in all cases 73

List of Figures
Figure 1-1. Differences between groups in mean NB during the feeding period 36
Figure 2-1. Differences between groups in mean serum albumin and serum prealbumin during the feeding period 55
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系統識別號 U0026-0208201523473800
論文名稱(中文) 利用環糊精與聚胺酸兩親分子形成之熱感型水膠
論文名稱(英文) Thermo-Responsive Supramolecular Hydrogels Formed by Cyclodextrin and Peptide Amphiphiles
校院名稱 成功大學
系所名稱(中) 化學工程學系
系所名稱(英) Department of Chemical Engineering
學年度 103
學期 2
出版年 104
研究生(中文) 許毓芸
學號 n36021329
學位類別 碩士
語文別 中文
口試日期 2015-07-06
論文頁數 73頁
口試委員 指導教授-詹正雄
口試委員-林睿哲
口試委員-王勝仕
口試委員-侯聖澍
口試委員-陳美瑾
關鍵字(中) 包嵌複合體
溫度應答
環糊精
聚胺基酸
兩親分子
關鍵字(英) inclusion complex
thermo-responsive
cyclodextrins
peptides
amphiphiles
學科別分類
中文摘要 本研究利用環糊精(cyclodextrins,CDs)與聚胺基酸兩親分子(peptide amphiphiles)形成包嵌複合體(inclusion complex),製備溫度敏感型的水膠。藉由改變疏水鏈長度、胺基酸單體的種類、環糊精的種類、聚胺基酸/環糊精比例與聚胺基酸濃度以調控水膠的物理性質,如轉化溫度與成膠濃度等,並藉由各種實驗分析探討聚胺基酸與環糊精的分子間作用力,以及分子間的排列方式。本實驗以具有直碳鏈的一級胺作為起始劑,對胺基酸進行開環聚合反應,得到不同親疏水鏈段長度的聚胺基酸兩親分子,其聚合度約為20,接近聚合反應時的單體與起始劑的比值。研究水膠的凝膠-溶膠轉化溫度與水膠濃度的關係,以流變儀驗證水膠性質與溫度的關係,並比較不同水膠的黏彈性質。為了研究聚胺基酸與環糊精之間的疏水作用力與氫鍵作用力,以及兩者形成水膠後在空間中的排列方式,以1H NMR證明環糊精可串入碳鏈而形成包嵌複合體,並以XRD確認環糊精的分子間氫鍵與結晶性質。由FTIR分析聚胺基酸鏈在加入環糊精後的二級結構變化,其中C16Thr20與β-CD形成的水膠有較高比例的分子間氫鍵,具有較高的凝膠-溶膠轉化溫度。以SAXS分析聚胺基酸與環糊精的堆疊方式,其中C12Thr20與α-CD形成雙層(lamellar)堆疊,C16Thr20與β-CD則無特定的堆疊方式。
英文摘要 In this study, we reported the preparation of thermo-responsive supramolecular hydrogels via inclusion complexation between cyclodextrins (CDs) and peptide amphiphiles (PAs). Alkylamine were used as the macroinitiator of ring-opening polymerization (ROP) to synthesize PAs. CDs threaded onto alkyl chain and form inclusion complex. The network structure of hydrogels was composed of hydrophobic interactions between alkyl chain and inner cavity of CDs, hydrogen bonding between CDs and side chain of PAs. The gel-sol transition temperature and gelation concentration were tuned by alkyl chain length, type of amino acids and CDs, the molar ratio of CDs and PAs. The secondary structure of peptides was mainly random coil. C12Thr20+α-CD hydrogels formed lamellar packing and the one bilayer thickness decreased with increasing temperature. The intelligent hydrogels could be promising in tissue engineering.
論文目次 摘要 I
Extended Abstract II
誌謝 VIII
目錄 X
表目錄 XIV
圖目錄 XV
第一章 緒論 1
1.1 前言 1
1.1.1 生醫材料 1
1.1.2 聚胺基酸與蛋白質 2
1.1.3 水膠 3
1.1.4 包嵌複合體 3
1.2 研究動機與目的 4
第二章 文獻回顧 5
2.1 聚胺基酸 5
2.1.1 蛋白質與胺基酸 5
2.1.2 胺基酸之基本性質 5
2.1.3 蛋白質之結構 7
2.1.4 胺基酸之聚合 10
2.2 水膠 12
2.2.1 水膠之定義 12
2.2.2 水膠之種類 12
2.2.3 聚胺基酸水膠應用於生物材料 14
2.3 包嵌複合體 15
2.3.1 環糊精發展史 15
2.3.2 主體-客體化學 16
2.3.3 環糊精之包嵌複合作用 17
2.3.4 以環糊精製備智慧型水膠 19
第三章 實驗方法與步驟 21
3.1 實驗藥品 21
3.2 實驗儀器與原理 23
3.2.1 液態核磁共振儀 23
3.2.2 基質輔助雷射脫附游離飛行時間質譜儀 23
3.2.3 X光繞射儀 24
3.2.4 流變儀 24
3.2.5 傅立葉轉換紅外線光譜儀 24
3.2.6 小角度X光散射儀 26
3.3 聚胺基酸之合成 27
3.3.1 N-carboxylanhydrides (NCAs)開環聚合法 27
3.3.2 乾燥溶劑 27
3.3.3 Z-L-lysine、benzyl-L-glutamate NCAs之製備 27
3.3.4 N-Boc-O-benzyl-L-threonine NCAs之製備 28
3.3.5 以一級胺對NCAs開環合成聚胺基酸 29
3.3.6 去除聚胺基酸之保護基Z group 29
3.3.7 去除聚胺基酸之保護基benzyl group 29
3.4 超分子(supramolecular)水膠之性質測試 30
3.4.1 以聚胺基酸與環糊精製備水膠 30
3.4.2 聚胺基酸與環糊精之包嵌複合性質 30
3.4.3 包嵌複合體之結晶性質 31
3.4.4 凝膠-溶膠轉化溫度 31
3.4.5 超分子水膠之流變性質 31
3.4.6 聚胺基酸之二級結構 32
3.4.7 聚胺基酸之堆疊 32
第四章 結果與討論 33
4.1 聚胺基酸之合成分析 33
4.1.1 聚胺基酸之聚合度 33
4.1.2 聚胺基酸之分子量 38
4.2 聚胺基酸與環糊精之包嵌複合性質 41
4.2.1 超分子水膠之形成 41
4.2.2 聚胺基酸與環糊精之疏水作用力 45
4.2.3 包嵌複合體之結晶性質 48
4.3 超分子水膠之物理性質 51
4.3.1 水膠濃度與溫度之關係 51
4.3.2 超分子水膠之流變性質 52
4.4 超分子水膠之結構 55
4.4.1 聚胺基酸之二級結構 55
4.4.2 聚胺基酸之堆疊 57
第五章 結論 61
參考文獻 62
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系統識別號 U0026-0409201221542600
論文名稱(中文) 動脈硬化中離胺基氧化酶和細胞外基質的交互作用
論文名稱(英文) Interplay between lysyl oxidase and extracellualr matrix homeostasis in arterial stiffness
校院名稱 成功大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Institute of Clinical Medicine
學年度 100
學期 2
出版年 101
研究生(中文) 張育慈
學號 S96991025
學位類別 碩士
語文別 英文
口試日期 2012-07-31
論文頁數 58頁
口試委員 指導教授-蔡曜聲
口試委員-蔡佩珍
口試委員-吳華林
口試委員-江美治
關鍵字(中) 動脈硬化
離胺基氧化酶
血管收縮素II
血管旁脂肪
關鍵字(英) Aortic stiffness
Lysyl oxidase
Angiotensin II
PVAT
學科別分類
中文摘要 肥胖是造成中風,心肌梗塞,心臟衰竭…等這些心血管疾病的危險因子。之前的研究指出,利用脈波傳導速度當作動脈硬化的指標,發現肥胖的病人脈波傳導速度是明顯增加的,表示肥胖的病人發生動脈硬化的機率是增加的。但是對於為什麼肥胖引起動脈硬化的機制目前還尚未清楚。彈性蛋白和膠原蛋白是在血管中影響動脈的彈性以及硬度的蛋白質,而要使這兩種蛋白質具有正常功能則需要離胺基氧化酶的催化。在我們之前的研究中發現,有基因缺陷導致肥胖的老鼠中離胺基氧化酶是明顯下降的,但是目前我們對於為什麼離胺基氧化酶會下降的原因還不清楚。並且我們觀察到在肥胖的老鼠中,其血管旁脂肪組織明顯的比起正常的老鼠厚。在之前的文獻指出血管旁的脂肪細胞可以利用旁分泌來調控血管的功能。因此,我們假設在肥胖老鼠中,血管旁的脂肪組織所分泌出來的物質會使離胺基氧化酶下降,導致細胞外基質平衡受到破壞,進而使動脈硬化的發生。首先,我們用膠原蛋白膠收縮分析法可以知道利用離胺基氧化酶的抑制劑BAPN降低離胺基氧化酶的活性會導致膠原蛋白膠的縮小。接下來我們在老鼠上給予BAPN以觀察是否會有動脈硬化的發生。我們發現高濃度的BAPN會導致動脈硬化並且增加彈性蛋白的斷點。接下來,我們探討在血管發生結構重塑的過程中離胺基氧化酶所扮演的角色,我們給予同時給予血管收縮素和BAPN。抑制離胺基氧化酶會導致更嚴重的血管重塑以及造成動脈硬化和血管瘤的發生。接下來我們探討,脂肪細胞和血管旁的脂肪細胞所分泌的物質是否會影響到血管平滑肌細胞離胺基氧化酶的活性。因此我們在血管平滑肌細胞上給予3T3-L1所分化的脂肪細胞以及從肥胖老鼠的血管旁脂肪所收集下來的培養液。發現脂肪細胞所分泌的物質會降低離胺基氧化酶的活性。我們也發現給予腫瘤壞死因子增加發炎反應以及雙氧水增加氧化壓力後發現離胺基氧化酶的活性也明顯地被抑制。從我們的結果可以發現在肥胖的老鼠中,血管旁細胞所分泌的物質可能是造成離胺基氧化酶活性下降的原因,因此破壞了細胞外基質的平衡,造成動脈硬化的發生。
英文摘要 Obesity is a significantly independent predictor of cardiovascular risk factor, including stroke, myocardial infarction, heart failure, and overall mortality. Recently, it has been reported that aortic stiffness, reflected by increasing pulse wave velocity (PWV), is increased in obesity. However, the mechanism of obesity-induced aortic stiffness is still unclear. Two main extracellular proteins, collagen and elastin, regulate vascular stability and compliance. However, the function of collagen and elastin are dependent on catalytic activity of lysyl oxidase (LOX). While we have shown that LOX was downregulated in obese ob/ob mice, the mechanism underlying this phenomenon remains unclear. We also observed that the aorta of ob/ob mice is surrounded by abundant perivascular adipose tissue (PVAT). It has been reported that PVAT regulates vascular function through paracrine effects. Therefore, we hypothesized that LOX downregulation, caused by the factors secreted from PVAT, leads to aortic stiffening in obese mice. To test this hypothesis, we used collagen gel contraction assay to determine whether downregulation of LOX by BAPN affected cross-linking. We found that inhibition of LOX activity led to smaller collagen gel. To investigate whether downregulation of LOX triggered aortic stiffness, we administrated mice with BAPN. We found that high dose of BAPN (300, and 600 mg/kg/day) induced aortic stiffness and elastin fragmentation in mice. Furthermore, to verify the role of LOX during aortic remodeling, we administrated BAPN under infusion of angiotensin II. Short-term treatment with BAPN alone (3 weeks) or angiotensin II alone (4 weeks) did not induce aortic stiffness. However, co-treatment of BAPN and angiotensin II increased aortic stiffening and the incidence of aneurysm. Therefore, these results suggested that BAPN promoted angiotensin II-induced aortic remodeling and stiffening. We further studied whether adipocyte-secreted factors downregulated LOX activity. Vascular smooth muscle cells (VSMCs) were treated with conditioned medium that collected from PVAT of ob/ob mice and differentiated 3T3-L1 adipocytes. We found that treatment of 3T3-L1 adipocytes and PVAT conditioned medium significantly downregulated LOX activity in a dose-dependent manner. Finally, we found that LOX activity was significantly decreased in VSMCs by treating them with TNF-α and H2O2. These data suggest that downregulation of LOX, possibly through paracrine effect of PVAT, impaired extracellular matrix homeostasis and triggered aortic stiffness in ob/ob mice
論文目次 INTRODUCTION 1
Structure of aorta 1
Aortic stiffness 2
Collagen and elastin 2
Perivascular adipose tissue (PVAT) 3
Lysyl oxidase 4
Role of LOX in diseases 5
β-aminopropionitrile (BAPN) 5
Angiotensin II in vascular disease 6
Significance 7
MATERIAL AND METHOD 8
Culture of A7r5 8
Culture of 3T3-L1 8
Differentiation of 3T3-L1 8
Collection of 3T3-L1 conditioned medium and treatment of A7r5 9
Collection of PVAT conditioned medium 9
Freezing cells 9
LOX activity 10
Collagen gel 10
Protein extraction 11
Western blot 11
Administration of β-aminopropionitrile (BAPN) 11
Measurement of pulse wave velocity 12
Measurement of blood pressure 12
Elastolytic activity 13
Tissue harvesting 13
Histology and elastic fiber stain 13
Data analysis 14
RESULT 15
Inhibition of LOX affected extracellular matrix cross-linking 15
Downregulation of LOX activity induced aortic stiffness 15
BAPN triggered elastin fragmentation and media thickening 17
BAPN promoted angiotensin II–induced aortic remodeling and stiffening 18
Co-treatment of BAPN and angiotensin II triggered media thickening 19
Adipocyte-secreted factors downregulated LOX activity in the vascular smooth muscle cell 19
Inflammatory cytokine and ROS reduced LOX activity in vascular smooth muscle cell 20
DISCUSSION 22
REFERENCE 30
FIGURE 40
Figure 1. Downregulation of LOX activity led to smaller collagen gel. 40
Figure2. Administration of low dose BAPN did not triggered aortic stiffness. 41
Figure 3. Long-term administration of high dose of BAPN triggered aortic stiffness. 42
Figure 4. BAPN did not affect the synthesis of extracellular matrix proteins. 43
Figure 5. Downregulation of LOX did not affect the thoracic aorta. 45
Figure 6. BAPN triggered elastin fragmentation in the thoracic aorta. 46
Figure 7. BAPN did not affect collagen content in the thoracic aorta. 47
Figure 8. Inhibition of LOX significantly affected the abdominal aorta. 49
Figure 9. BAPN triggered elastin fragmentation in abdominal aorta. 50
Figure 10. BAPN did not induce collagen content in the abdominal aorta. 51
Figure 11. The group of Angiotensin II alone and co-treatment did not increase blood pressure 52
Figure 12. BAPN promoted angiotensin II-induced aortic remodeling. 53
Figure 13. BAPN promoted angiotensin II-induced aortic aneurysm. 54
Fig 14. Co-treatment with BAPN and angiotensin II appeared not to affect the thoracic aorta. 55
Fig 15. Co-treatment with BAPN and angiotensin II increased aortic wall area in abdominal aortas. 56
Figure 16. The factors secreted from differentiated 3T3-L1 adipocyte and PVAT downregulated LOX activity in A7r5 VSMC and led to smaller collagen gel. 57
Figure 17. TNF-α and H2O2 downregulated LOX activity of A7r5. 58
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系統識別號 U0026-0812200915280173
論文名稱(中文) 芝麻油對健大黴素所引起大鼠腎臟傷害的保護作用之探討
論文名稱(英文) Effect of sesame oil on gentamicin-induced renal injury in rats
校院名稱 成功大學
系所名稱(中) 環境醫學研究所
系所名稱(英) Institute of Environmental and Occupational Health
學年度 97
學期 2
出版年 98
研究生(中文) 劉傳騰
學號 s7696108
學位類別 碩士
語文別 中文
口試日期 2009-06-29
論文頁數 58頁
口試委員 口試委員-許德榮
口試委員-邱元佑
召集委員-林永明
指導教授-劉明毅
關鍵字(中) 超陰氧離子
反應性氧族
腎毒性
氧化壓力
健大黴素
胺基配醣體
關鍵字(英) nephrotoxicity
oxidative stress
gentamicin
aminoglycoside
superoxide anion
reactive oxygen species
學科別分類
中文摘要 健大黴素是一種胺基多醣體的抗生素,主要被用來對抗格蘭氏陰性菌的感染;然而,在臨床上常會產生腎毒性等副作用。許多文獻指出,氧化性壓力可能與健大黴素所造成的腎毒性有關。芝麻油,在東方國家被認為是適合食用的油脂類及健康食品,具有高度的穩定性及抗氧化能力,然而芝麻油對健大黴素所引起大鼠腎臟傷害的作用仍不清楚。因此,本研究目的為探討芝麻油對健大黴素所引起大鼠腎臟傷害的影響。實驗動物給予健大黴素(100 mg/kg/d, sc),並同時給予不同劑量芝麻油(0 ~ 8 ml/kg/d, oral),連續七天後,收集動物尿液、血液及腎臟組織。結果顯示,芝麻油能具有改善腎小管組織壞死及肌酸酐清除率、降低血中尿素氮與肌酸酐含量、降低腎臟脂質過氧化情形、減少羥自由基含量、降低超氧陰離子及一氧化氮的生成、減少黃嘌呤氧化及誘導型一氧化氮合成的表現。在治療效應的實驗中,單一劑量的芝麻油(0 ~ 8 ml/kg, oral),也能夠有效的減輕血中尿素氮及肌酸酐含量、減輕腎臟脂質過氧化、減少過氧亞硝基物、降低超氧陰離子及一氧化氮的生成。綜合以上結果,不論每日給予共七劑或只給予單一劑量的芝麻油皆可有效保護大鼠經健大黴素所誘發的腎臟傷害,其機制可能經由降低氧化壓力的產生,進而減緩腎臟之氧化性傷害。
英文摘要 Objective: The aim of the present study was to assess the effect of sesame oil on gentamicin-induced renal damage in rats.
Design: ( i ) Rat renal injury was induced by gentamicin (100 mg/kg/d, sc), and sesame oil (ranging 0 to 8 ml/kg/d, orally) was given simultaneously for one week. ( ii ) In therapeutic experiment, a single dose of sesame oil (ranging 0 to 8 ml/kg, orally) was given after gentamicin administration. Serum renal function as well as renal lipid peroxidation, hydroxyl radical, peroxynitrite, superoxide anion, nitric oxide, xanthine oxidase, and nitric oxide synthase were determined 8 days post-treatment.
Results: ( i ) Daily supplement of sesame oil (a) attenuated renal tubular necrosis and creatinine clearance, (b) inhibited serum blood urea nitrogen,
creatinine levels, renal lipid peroxidation, hydroxyl radical, superoxide anion, nitric oxide, xanthine oxidase, and nitric oxide synthase in gentamicin-treated rats.( ii ) A single dose of sesame oil ameliorated serum blood urea nitrogen, creatinine levels and inhibited renal lipid peroxidation, peroxynitrite, superoxide anion and nitrite in gentamicin-treated rats.
Conclusion: Sesame oil might attenuate gentamicin-induced renal injury by inhibiting oxidative stress in rats, and might be a useful therapeutic agent.
論文目次 致謝 2
中文摘要 3
英文摘要 4
圖 目 錄 5
附 錄 6
研究背景 7~12
研究目的 13
材料與方法 14-23
實驗結果 24-28
實驗結果討論 29-32
研究結論 33
參考文獻 34-41
實驗圖表 42-54
研究附錄 55-57
論文著作 58
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系統識別號 U0026-0907201214053300
論文名稱(中文) 水中數種藍綠菌與藻類在氯胺消毒程序中亞硝基二甲胺之生成潛能研究
論文名稱(英文) Formation Potential of N-nitrosodimethylamine for Several Algae and Cyanobacteria during Chloramination
校院名稱 成功大學
系所名稱(中) 環境工程學系碩博士班
系所名稱(英) Department of Environmental Engineering
學年度 100
學期 2
出版年 101
研究生(中文) 曾靖樺
學號 p56994187
學位類別 碩士
語文別 中文
口試日期 2012-06-27
論文頁數 100頁
口試委員 指導教授-林財富
口試委員-葉宣顯
口試委員-蔣本基
口試委員-王根樹
關鍵字(中) 藍綠菌
藻類
胺基酸
氯胺消毒
亞硝基二甲胺
生成潛能
關鍵字(英) Algae
Amino Acid
Chloramination
Cyanobacteria
Fomration Potential
N-nitrosodimethylamine (NDMA)
學科別分類
中文摘要 亞硝基二甲胺 (N-nitrosodimethylamine, NDMA) 於西元1989年首度於飲用水處理系統中被發現,由於具有高度致癌風險,成為國際關注之新興含氮消毒副產物。優養化原水含有高濃度藍綠菌/藻類存在,其細胞體與代謝物會藉由生物代謝與固氮作用增加天然水之溶解性有機氮 (Dissolved organic nitrogen, DON),這些DON可能會在消毒過程中與氯/氯胺作用產生NDMA,造成水質安全疑慮。因此,本研究探討原水中所含藍綠菌/藻類之細胞體與代謝物的NDMA生成潛能關係,最後並以胺基酸模擬DON,建立胺基酸與NDMA生成潛能關係。

本研究利用一氯胺作為氧化劑,改變不同氧化條件 (氧化劑接觸反應時間、氧化劑濃度、藻體細胞數、胺基酸溶液pH值、氯氮比例、含氮濃度及胺基酸種類),氧化數種水中常見之藍綠菌與藻類、主要胺基酸及天然水庫原水,瞭解水體經氯胺消毒過程中亞硝基二甲胺 (NDMA) 之生成潛能。並探討不同藍綠菌與藻類、胺基酸種類與天然水體之相關性與NDMA生成潛能推估之應用性。

結果顯示,藍綠菌 (柱孢藻、微囊藻、魚腥藻及針形藻) 與藻類 (小球藻與菱形藻) 皆為細胞體較代謝物具有較高之NDMA生成潛能。以菱形藻藻體之NDMA生成量為代謝物之5.3倍,差異最大,柱孢藻則次之。且隨氧化劑接觸反應時間、氧化劑量與細胞數量升高NDMA生成潛能增加。於含氮濃度0.05 mM胺基酸溶液氧化實驗中,一氯胺濃度為2.0 mM,僅絲胺酸 (Serine) 於反應後沒有測得NDMA,天冬胺酸 (Aspartic acid) 則生成最高量,達92.8 ng/L。研究中後續利用氧化細胞懸浮液與EOM之NDMA生成量,計算得藍綠菌/藻類細胞NDMA生成潛能、與天然水中細胞種類和數量,進一步推估天然水NDMA生成潛能。當水體屬優養程度時,則其計算值與實驗值相近;然當水體為普養程度時,則以直接氧化細胞體獲得之NDMA生成潛能,較接近水體NDMA生成潛能。

此研究結果有利於瞭解不同藍綠菌與藻類之NDMA生成潛能和生成機制探討,且可評估現行飲用水之藍綠菌相關警戒規範是否亦適用於NDMA生成潛能警戒,以保障飲用水水質安全。
英文摘要 N-nitrosamines have become an important issue in drinking water science since they were identified as disinfection by- products (DBPs) in chloraminated waters. N-nitrosodimethylamine (NDMA) is the most frequently detected member of nitrosamines in drinking water. The risk that the chemical poses to consumer health seems to be much higher than that from chlorinated DBPs. Although the first report that documented the presence of NDMA in water appeared in the 1970s. NDMA was widely discovered in many finishd waters at the beginning of the 21st centry. Although the monitoring and formation potential of NDMA has been extensively studied, the formation potential of cyanobacteria during chloramination has never been systematically investigated. In this study, formation potential of NDMA during chloramination of two algae, four cyanobacteria and two cyanobacteria-laden reservoir waters is investigated. In addition, chloramination of 17 amino acids were also studid for the formation potential of NDMA.
Experimental results indicated that NDMA formed from chloramination of cyanobacteria and algae cells is more than that from chloramination with extracellular organic matter associated with cyanobacteria and algae. In the system, higher oxidant dose, longer contact time, and more cell numbers would produce more NDMA. For chloramination of amino acid, NDMA was detected for almost all the cases, with highest concentration for aspartic acid (92.8 ng/L). The results of cyanobacteria and algae cells were linked to the formation protential for selected cyanobacteria-laden reservoir waters, through calculation of cell number in cyanobacteria-laden reservoir waters and formation protential of dominant species. The calculated results for NDMA formation are comparable with those from experiments, indicating that cyanobacteria and algae cells are important precursors of NDMA. The methodology developed may be used to estimate the risk associated with NDMA for the water with significant amounts of cyanobacteria and algae.
論文目次 摘要 I
Abstract III
誌謝 V
目錄 VII
表目錄 XI
圖目錄 XII

第一章 前言 1
1.1 研究緣起 1
1.2 研究目的 2
第二章 文獻回顧 3
2.1 消毒副產物 3
2.1.1 含碳消毒副產物 3
2.1.2 新興含氮消毒副產物 5
2.2 亞硝基二甲胺 6
2.2.1 NDMA物化特性 9
2.2.2 NDMA形成機制 10
2.2.3 NDMA管制標準 13
2.3 天然有機氮源 14
2.3.1 藍綠菌與藻類 15
2.3.2 胺基酸 18
2.3.3 有機氮源管制 20
第三章 實驗材料與方法 21
3.1 藍綠菌與藻類培養 22
3.1.1 藍綠菌與藻類來源 22
3.1.2 培養方法 24
3.2 細胞計數與生物體積量測 26
3.2.1 細胞計數方法 26
3.2.2 細胞生物體積計算 29
3.3 溶解性有機氮分析 30
3.4 NDMA生成潛能實驗 33
3.4.1 一氯胺配製 33
3.4.2 實驗前置準備 35
3.4.2.1 藍綠菌與藻類 35
3.4.2.2 胺基酸溶液 36
3.4.2.3 天然水 37
3.4.3 實驗步驟 39
3.5 NDMA分析 43
第四章 結果與討論 47
4.1 藍綠菌與藻類生成NDMA潛能影響 47
4.1.1 氧化劑接觸反應時間 47
4.1.2 氧化劑濃度 51
4.1.3 細胞體數目 60
4.2 藍綠菌與藻類生成NDMA剖析與應用 65
4.2.1 細胞單位生物體積 65
4.2.2 藍綠菌與藻類NDMA生成潛能剖析 66
4.2.3 氮類物質和DON含量與NDMA生成潛能剖析 71
4.2.4 天然水NDMA生成潛能 72
4.2.5 現行原水規範與NDMA生成潛能 74
4.3 胺基酸生成NDMA潛能影響 78
4.3.1 氧化劑接觸反應時間 78
4.3.2 溶液pH值 80
4.3.3 氯/氮比例 83
4.3.4 胺基酸種類與NDMA生成潛能關係 86
4.4 胺基酸 NDMA生成剖析與應用 89
4.4.1 細胞體胺基酸組成與NDMA生成潛能 89
4.4.2 天然水胺基酸組成NDMA生成潛能 92
第五章 結論與建議 93
5.1 結論 93
5.2 建議 94
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系統識別號 U0026-0909201414100600
論文名稱(中文) 含甘露醣之醣胜肽抗菌分子的前導合成研究
論文名稱(英文) Model studies toward the preparation of mannopeptimycins
校院名稱 成功大學
系所名稱(中) 化學系
系所名稱(英) Department of Chemistry
學年度 102
學期 2
出版年 103
研究生(中文) 郭亦詠
學號 L36014207
學位類別 碩士
語文別 中文
口試日期 2014-07-28
論文頁數 120頁
口試委員 指導教授-鄭偉杰
共同指導教授-黃福永
口試委員-張夢揚
關鍵字(中) 抗生素
胺基酸
醣肽分子
雙醣
關鍵字(英) antibiotics
amino acid
glycopeptide
disaccharide
學科別分類
中文摘要 Mannopeptimycins是一種醣肽分子的抗生素,對於抗藥性金黃色葡萄球菌(methicillin-resistant Staphylococcus aureus, MRSA) 和抗萬古黴素腸球菌 (vancomycin-resistant Enterococcus faecium, VRE)皆具有良好的抑制活性(MIC: 4-128 μg/mL)。可能藉由與Lipid II受質結合來抑制細胞壁生合成。由於此醣肽分子的全合成尚未發表,並且在先前片段合成的設計仍有修飾的空間,特別在兩個罕見的胺基酸:(2R, 3S, 4S)-hydroxyenduracididine和(2S, 3S, 4S)-hydroxyenduracididine的合成。因此本篇論文著重在這兩個具有三個連續的立體化學罕見胺基酸之合成設計:第一個罕見胺基酸(2R, 3S, 4S)-hydroxyenduracididine,利用對掌性內酯10為起始物,經由12個反應步驟可有效率得到目標分子,並且有良好的產率(7.2%)。第二個罕見胺基酸(2S, 3S, 4S)-hydroxyenduracididine,僅在二號碳位置的立體化學不同,可利用對掌性內酯11,藉由加入對甲苯磺酸吡啶鹽(pyridinium p-toluenesulfonate, PPTS)使其產生熱力學穩定的產物,改變二號碳位置的立體化學,再經由12個反應步驟得到目標分子,總產率3.2%。
在合成設計上,第一種合成設計,內酯藉由氫氧化鋰開環,再經過一系列的化學轉換形成具有多種官能基保護的亞芐基 (benzylidene)中間產物,但在亞芐基之選擇性開環較不順利,因此採用第二種合成設計,內酯藉由還原形成雙羥基產物,再經由一系列的化學轉換形成亞芐基中間產物,幸運地可利用TiCl4/NaCNBH3進行選擇性開環。另一個mannopeptimycin之片段─醣肽分子,利用D-甘露糖(D-mannose)為起始物,經由10步驟得到雙醣分子,並使其形成好的醣予體(donor),在BF3•OEt2催化下與Fmoc-D-tyrosine之羥基進行醣基化得到醣肽分子。
英文摘要 In this research, we aim at synthetic design of mannopeptimycin fragment including two unusual amino acids with three continuous stereocenters: (2R, 3S, 4S)-hydroxyenduracididine, (2S, 3S, 4S)-hydroxyenduracididine and D-tyrosine-linked glycan.
The first unusual amino acid, (2R, 3S, 4S)-hydroxyenduracididine, has been achieved in twelve steps from the known chiral lactone 10. The efficiency of the synthesis is demonstrated by the high overall yield of 7.2%. The other unusual amino acid, (2S, 3S, 4S)-hydroxyenduracididine, an epimer of the first one, has been synthesized in twelve steps from the chiral lactone 25 with overall yield of 3.2%. The chiral lactone 25, a thermodynamic product was prepared by adding pyridinium p-toluenesulfonate from lactone 11.
The key transformation of the synthetic route is selective benzylidene ring opening. Fortunately, the selective ring opening of benzylidene was obtained by using TiCl4/NaCNBH3.
The design of the other mannopeptimycin fragment, D-tyrosine-linked glycan, by preparing the disaccharide in ten steps from D-mannose. BF3•OEt2-catalyzed glycosylation was achieved with Fmoc-D-tyrosine to afford the D-tyrosine-linked glycan.
論文目次 目錄

摘要 I
Extended Abstract II
誌謝 VII
目錄 IX
圖目錄 XI
表目錄 XII
流程目錄 XIII
中英文對照表 XIV
簡稱語對照 XVI
第一章 緒論 1
1.1 簡介及背景 1
1.2 Mannopeptimycin主架構及分類 2
1.3細胞壁生合成過程及mannopeptimycin作用位置 3
1.4 Mannopeptimycin片段合成介紹 5
1.4.1 醣肽分子及雙醣分子的合成 5
1.4.2 Hydroxyenduracididines的合成 6
1.5 研究目的與動機 8
第二章 結果與討論 9
2.1 Mannopeptimycin ε類似物之合成設計 9
2.2 Hydroxyenduracididines (1a, 2)的合成 11
2.2.1 Hydroxyenduracididine (1a)逆合成分析 11
2.2.2 中間產物10的製備 13
2.2.3中間產物5的製備及選擇性開環 14
2.2.4 化合物1a之合成路徑 17
2.2.5 化合物28和2之合成 22
2.2.6 化合物2之合成 25
2.3 醣肽化合物3之合成 26
2.3.1 化合物38的製備 26
2.3.2 化合物3之合成 27
2.4 光譜討論 28
2.5 總結 30
第三章 實驗部分 31
3.1 實驗藥品 31
3.2 實驗儀器 31
3.3 實驗步驟與光譜數據 32
參考文獻 57
附錄 62
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系統識別號 U0026-1007201300373600
論文名稱(中文) 醣與硫辛酸修飾聚賴胺酸與聚丙烯酸自組裝之還原及酸鹼應答複合高分子
論文名稱(英文) Redox- and pH-responsive complex micelles assembled from saccharide- and lipoic acid-modified poly(L-lysine) and poly(acrylic acid)
校院名稱 成功大學
系所名稱(中) 化學工程學系碩博士班
系所名稱(英) Department of Chemical Engineering
學年度 101
學期 2
出版年 102
研究生(中文) 謝秉倫
學號 n36004327
學位類別 碩士
語文別 中文
口試日期 2013-06-25
論文頁數 81頁
口試委員 指導教授-詹正雄
口試委員-王勝仕
口試委員-林宏殷
口試委員-張鑑祥
口試委員-吳文中
關鍵字(中) 雙環境應答
聚胺基酸
聚電解質複合粒子
藥物傳輸
醣修飾
關鍵字(英) dual-response
polypeptide
polyelectrolyte
complex particles
galactose
學科別分類
中文摘要 本研究製備對酸鹼值與還原環境應答,且具有細胞標靶功能的聚電解質複合 (polyelectrolyte complex) (PEC) 粒子,組成PEC粒子的聚電解質分別為:帶正電的醣修飾聚賴胺酸接枝硫辛酸(lactobionolatone modified poly(L-lysine)-graft-lipoic acid) (PLL-g-Lipo-g-Lac)與負電的聚丙烯酸(poly (acrylic acid)) (PAA),混合形成PEC粒子帶電外層由過量的聚電解質與雙醣基團組成,其中PLL-g-Lipo-g-Lac是由具保護基的賴胺酸形成N-carboxyanhydrides (NCAs)經開環聚合(ring-opening polymerization) (ROP)合成poly(Z-L-lysine) (PZLL),PZLL去除保護基後經過硫辛酸修飾與醣修飾後得到PLL-g-Lipo-g-Lac,不同分子量PLL與硫辛酸和醣接枝的比例則由超導核磁共振光譜儀(Nuclear Magnetic Resonance Spectrophotometer)(NMR)鑑定,不同溶液條件下製得PEC粒子的粒徑、Zeta-potential、形態與結構,分別由動態光散射分析儀(dynamic light scattering) (DLS)、Zeta 電位分析儀、穿透式電子顯微鏡(transmission electron microscopy) (TEM) 與圓二色光譜儀(circular dichroism) (CD)分析。藉由調控組成的聚電解質比例、分子量與接枝比例,PEC粒子可以帶有正或負的不同電性,形成的粒徑在50到200奈米之間。實驗結果顯示,PEC粒子能對環境的酸鹼度與還原特性做出應答,藉由加入具專一性的凝集素與PEC粒子外層的醣基結合,證明PEC粒子具有生物辨識功能,再配合PEC具有的雙環境應答功能,,這些PEC粒子具有應用藥物傳輸與標靶治療等生醫領域的潛力。
英文摘要 In this study, novel pH and redox-responsive, cellular targeting polyelectrolyte complex (PEC) particles were developed. By mixing the polycation and polyanion, lactobionolatone modified poly(L-lysine)-graft-lipoic acid (PLL-g-Lipo-g-Lac) and poly acrylic acid (PAA), PEC particles can be formed with excess polyelectrolyte and saccharide group on the corona. PLL polypeptides were prepared by synthesizing poly(Z-L-lysine) (PZLL) via ring-opening polymerization (ROP) from Z-L-lysine N-carboxyanhydrides (NCAs) and subsequently removing the protecting Z group. PLL-g-Lipo-g-Lac graft copolypeptides with different polypeptide chain lengths and grafting degrees of lipoic acid were synthesized. The grafting degrees of lipoic acid and saccharide group were determined by Nuclear Magnetic Resonance Spectrophotometer (NMR). The size, charge, chain conformation, and morphology of the as-prepared PEC particles at different solution conditions were characterized by dynamic light scattering (DLS), aqueous electrophoresis, transmission electron microscopy (TEM) and circular dichroism (CD) measurements. The positively and negatively charged PEC particles with size ranged between 50 and 200 nm can be prepared by varying polymer chain length, grafting degree, and composition. The experimental data revealed that these PEC particles can response to the pH and oxidation/reduction changes in the environment. The selective lectin binding experiments confirmed that the galactose units on the particles can be used in biorecognition applications. Combining their dual-response and liver cell targeting ability, these PEC particles could be useful in biomedical fields such as drug delivery.
論文目次 總目錄
第一章 緒論 1
第二章 文獻回顧 3
2.1 聚胺基酸 3
2.1.1 聚胺基酸的結構與特性 3
2.1.2 胺基酸的聚合 9
2.2 雙親性高分子自組裝 13
2.2.1 自組裝行為介紹 13
2.2.2 自組裝高分子介紹 14
2.3 奈米粒子藥物載體 16
2.3.1 聚胺基酸做為藥物載體 18
2.3.2 奈米粒子藥物載體的設計 19
2.4 環境應答 21
2.4.1 酸鹼應答 21
2.4.2 溫度應答 23
2.4.3 還原應答 23
2.5 聚電解質複合材料 24
2.6 雙功能性應答系統 25
第三章 實驗方法與步驟 27
3.1 實驗藥品 27
3.2 實驗儀器與原理 29
3.2.1 凝膠滲透層析儀 29
3.2.2 核磁共振光譜儀 30
3.2.3 動態光散射偵測儀 31
3.2.4 Zeta電位分析儀 33
3.2.5 圓二色光譜儀 34
3.2.6 紫外線/可見光光譜儀 36
3.2.7 穿透式電子顯微鏡 36
3.3 實驗方法 37
3.3.1 聚胺基酸高分子之合成 37
3.3.2 聚胺基酸接枝及醣修飾 39
3.3.3 聚丙烯酸之合成 40
3.3.4 聚胺基酸與聚丙烯酸PEC粒子的製備 41
第四章 結果與討論 42
4.1 材料的合成與鑑定 42
4.1.1 聚賴胺酸高分子的合成、接枝與修飾 42
4.1.2 聚賴胺酸接枝硫辛酸之結構鑑定 44
4.1.3 聚丙烯酸的合成與鑑定 46
4.2 聚電解質PEC粒子的製備與鑑定 47
4.3 聚賴胺酸接枝高分子的雙醣修飾 51
4.4 醣修飾聚胺基酸高分子複合粒子功能性分析 56
4.4.1 雙醣修飾之活性測試 56
4.4.2 酸鹼應答 57
4.4.3 還原應答 59
4.4.4 PEC粒子的結構與形態鑑定 61
第五章 結論 65
第六章 參考文獻 66
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系統識別號 U0026-1108201409500400
論文名稱(中文) 利用金屬離子-聚電解質自組裝粒子為模板製備交聯之聚胺酸高分子中空奈米球及二氧化矽/聚胺酸高分子複合奈米殼
論文名稱(英文) Preparation of Cross-Linked Polypeptide Nanocages and Silica/Polypeptide Hybrid Nanoshells Templated by Metal ion-Polyelectrolyte Assemblies
校院名稱 成功大學
系所名稱(中) 化學工程學系
系所名稱(英) Department of Chemical Engineering
學年度 102
學期 2
出版年 103
研究生(中文) 劉思廷
學號 N36014495
學位類別 碩士
語文別 中文
口試日期 2014-07-02
論文頁數 85頁
口試委員 指導教授-詹正雄
口試委員-張鑑祥
口試委員-侯聖澍
口試委員-林宏殷
關鍵字(中) 中空奈米球
奈米殼
二氧化矽
氧化還原/酸鹼應答
藥物傳輸
交聯
聚胺基酸
關鍵字(英) nanocage
nanoshell
silica
redox/pH responsiveness
drug delivery
cross-linked
polypeptide
學科別分類
中文摘要 本研究中,藉由金屬離子及聚電解質透過靜電作用力形成自組裝結構為模板,製備聚胺基酸交聯微胞、聚胺酸交聯中空奈米球及二氧化矽/聚胺酸複合奈米殼,此複合微胞是由聚二乙烯三胺五乙酸與胱胺高分子(poly (diethylenetriaminepentaacetic dianhydride-co-cystamine), poly(DTPA-co- cystm))、金屬離子及聚乙二醇嵌段聚賴胺酸高分子(PEG-b-poly(L-lysine), PEG-b-PLL)所組成,具有氧化還原及酸鹼雙重應答之性質。複合微胞之尺寸大小可藉由改變莫耳比例、金屬離子或是製程來調控。並以京尼平(genipin)交聯聚胺基酸複合微胞,使其在稀釋及溶液環境條件改變時,結構不易崩解。藉由控制京尼平與胺基(NH2)之比例,可得到不同交聯比例之複合微胞。聚胺基酸交聯中空奈米球可利用榖胱甘肽(glutathione, GSH)切斷雙硫鍵,移除聚二乙烯三胺五乙酸與胱胺高分子得到。另外,利用聚胺基酸交聯中空奈米球交聯複合微胞為模板沉析二氧化矽,可形成二氧化矽/聚胺基酸複合奈米殼,此材料可應用於生物醫學領域。此外,奈米複合微胞可包覆抗癌藥物阿黴素(Doxrubicin, DOX),並藉由酸鹼值的降低或雙硫鍵的斷裂進行藥物釋放,此具酸鹼及氧化還原雙重應答之複合微胞將具有潛力成為藥物載體。
英文摘要 SUMMARY
In this study, we report the preparation of cross-linked polypeptide complex micelles, cross-linked polypeptide nanocages, and silica/polypeptide hybrid nanoshells templated by metal ion-polyelectrolyte assemblies via electrostatic interaction. The complex micelles composed of poly(diethylenetriaminepentaacetic dianhydride-co-cystamine) (poly(DTPA-co-cysam)), metal ions, and PEG-b-poly (L-lysine) (PEG-b-PLL) exhibited redox/pH dual-responsive property. Their sizes can be tuned by varying the molar ratio, metal ion species, or synthesis process. These complex micelles can be cross-linked by genipin to prevent dissociation upon dilution. The cross-linked polypeptide nanocages can be prepared and used as template to form silica/polypeptide hybrid nanoshells. Furthermore, these cross-linked micelles were employed for doxorubicin (DOX) encapsulation and it was found that the DOX release can be triggered by redox/pH dual-responsiveness. These complex micelles could be promising as drug carriers.
Keywords : nanocage, nanoshell, silica, redox/pH responsiveness, drug delivery, cross-linked, polypeptide
論文目次 摘要 I
Extended Abstract II
目錄 X
表目錄 XIV
圖目錄 XV
第一章 緒論 1
1.1 前言 1
1.2 研究動機與目的 2
第二章 文獻回顧 4
2.1 聚胺基酸 4
2.1.1 胺基酸之簡介 4
2.1.2 胺基酸之聚合 5
2.2 聚電解質複合粒子 7
2.2.1 聚電解質複合粒子之簡介 7
2.2.2 聚電解質複合粒子之分類 7
2.3交聯 9
2.4 中空奈米球(hollow nanosphere) 13
2.5 生物材料與礦化作用 14
2.6 奈米載體 16
2.6.1 奈米載體簡介 16
2.6.2 奈米載體之生物訊號應答 16
第三章 實驗方法與儀器設備 23
3.1 實驗藥品 23
3.2聚乙二醇與聚賴胺酸嵌段高分子(PEG-b-PLL)和聚二乙烯三胺五乙酸與胱胺高分子poly(DTPA-co-cysam)之合成 25
3.2.1溶劑之純化 25
3.2.2 N-carboxyanhydrides (NCAs)的製備 25
3.2.3 利用一級胺對NCAs開環合成聚胺基酸 26
3.2.4 去除聚胺基酸之保護基 26
3.2.5合成聚二乙烯三胺五乙酸與胱胺嵌段共聚物poly(DTPA-co-cysam) 27
3.3複合粒子之製備 27
3.3.1聚電解質金屬複合粒子之製備 27
3.3.2以京尼平交聯複合粒子 28
3.3.3以GSH製備中空複合粒子 29
3.3.4 製備二氧化矽/聚胺基酸複合奈米殼 29
3.4藥物包覆與釋放 29
3.5特性分析與性質測試 30
3.5.1液態核磁共振儀 30
3.5.2凝膠滲透層析儀 31
3.5.3動態光散射偵測儀 32
3.5.4穿透式電子顯微鏡 35
3.5.5紅外線光譜儀 36
3.5.6圓二色光譜儀 37
3.5.7紫外光/可見光光譜儀 38
3.5.8 SAXS 39
第四章 結果與討論 41
4.1聚乙二醇與聚賴胺酸嵌段高分子(PEG-b-PLL)和聚二乙烯三胺五乙酸與胱胺高分子poly(DTPA-co-cysam)之合成與分析 41
4.1.1聚乙二醇與聚賴胺酸嵌段高分子之製備與分析 41
4.1.2聚二乙烯三胺五乙酸與胱胺高分子poly(DTPA-co-cysam)之製備與分析 43
4.2 PEG-b-PLL/poly(DTPA-co-cysam)/metal ions奈米複合粒子之製備及分析 46
4.2.1 PEG-b-PLL/poly(DTPA-co-cysam)/metal ions奈米複合粒子之製備 46
4.2.2 PEG-b-PLL/poly(DTPA-co-cysam)/Fe3+(Al3+)奈米複合粒子之分析 48
4.3 PEG-b-PLL/poly(DTPA-co-cysam)/metal ions奈米複合微胞製備交聯之中空奈米球及二氧化矽/聚胺酸複合奈米殼 55
4.3.1交聯複合粒子之製備及粒徑分析 55
4.3.2利用榖胱甘肽(GSH)製備交聯之中空奈米球及分析 60
4.3.3二氧化矽/聚胺酸複合奈米殼之製備及分析 65
4.4 PEG-b-PLL/ poly(DTPA-co-cysam)/metal ions奈米複合粒子作為藥物載體之應用 68
4.4.1製備裝載藥物之複合粒子及其分析 68
4.4.2複合粒子裝載藥物後之釋放實驗及分析 70
第五章 結論 72
第六章 參考文獻 73
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系統識別號 U0026-1307201018515900
論文名稱(中文) 結合層層自組裝與模板法合成可控制奈米結構無機氧化物材料
論文名稱(英文) Synthesis of Controllable Oxide Nanostructures via Layer-by-Layer Assembly and Template-Directed Approaches
校院名稱 成功大學
系所名稱(中) 化學工程學系碩博士班
系所名稱(英) Department of Chemical Engineering
學年度 98
學期 2
出版年 99
研究生(中文) 莊子函
學號 n3697144
學位類別 碩士
語文別 中文
口試日期 2010-07-01
論文頁數 131頁
口試委員 指導教授-詹正雄
口試委員-陳東煌
口試委員-莊怡哲
口試委員-羅介聰
關鍵字(中) 層層自組裝
多孔性二氧化矽
聚胺基酸
金奈米粒子
中空管
觸媒降解
二級結構
關鍵字(英) layer-by-layer
porous silica
polypeptide
gold nanoparticles
hollow tube
secondary structure
學科別分類
中文摘要 這篇論文裡,我們製備出擁有奈米結構的有機/無機組合式複合材料並可合成出具有中空管狀的無機氧化物,二氧化矽和二氧化鈦,藉由模板法和不同的合成策略進行生物仿生的礦化作用,控制實驗的條件來改變材料形狀、尺寸大小、孔隙度和孔洞結構。我們利用合成材料聚胺基酸poly-L-lysine和poly-L-glutamic acid的物理性質、化學特性、合成途徑層層自組裝的過程和實驗溶液的變化,藉由這些因素來控制並影響形狀和尺寸大小,而聚胺基酸的二級結構是決定孔洞結構和孔隙度的重要因素之一,聚胺基酸高分子間的交互作用、不同的溶液狀態下都可能會觸發二級結構的生成。另外,結合金奈米粒子和多孔性二氧化矽形成的複合材料,可在相同的製程下獲得,同樣藉由控制實驗的條件和聚胺基酸的物理化學特性不僅影響二氧化矽材料同時也影響金奈米粒子生成的還原性、顆粒大小和分散性,達到高可控制性的複合材料。最後針對金/二氧化矽材料和二氧化鈦相關的觸媒降解應用,證明可藉由材料控制性來達到高觸媒活性。我們提供一種多用途性的技術合成奈米級的無機氧化物材料,材料都能表現出獨特的良好特性,並且能應用在許多相關領域裡,例如:藥物傳輸、包覆材料和奈米裝置等等。
英文摘要 Here we report the preparation of hollow nanostructured organic/inorganic composite tube and inorganic oxide materials that are silica and titanium with controllable shape, size, porosity, and pore architecture using a combination of different template-directed synthesis strategies, layer-by-layer assembly and followed by biomimetic mineralization. The size and shape of asynthesized materials can be controlled by polypeptide chemistry, template-directed approaches and solution conditions. In addition, the porosity and pore architecture of as-synthesized materials can be controlled by polypeptide chemistry, polypeptide secondary structures, the association of different polypeptides, and solution conditions. In the same procedure, we also proposed to use polypeptide macromolecular assemblies as mediating agents and templates for porous silica mineralization and directed growth of gold nanoparticle. The reported strategies provide simple and versatile techniques to synthesize oxide based nanomaterials at benign conditions. The results show that these materials have unique properties of relevance to numerous applications including drug delivery, nanodevices and encapsulation.
論文目次 目錄
摘要 I
Abstract II
誌謝 III
目錄 IV
表目錄 VIII
圖目錄 IX
第一章 緒論 1
1.1 前言 1
1.1.1 奈米複合材料 1
1.1.2 中空管的發展 2
1.3 生物材料 3
1.3.1 材料仿生學 3
1.3.2 自然材料與礦化作用的發展 4
1.3.3 聚胺基酸 5
1.3.4 聚賴胺酸和二級結構 7
1.4 研究動機與目的 9
第二章 文獻回顧 11
2.1 管狀形無機材料的製備技術與方法 11
2.1.1 模板法 12
2.1.2 層層自組裝薄膜技術 14
2.1.3 模板法結合層層自組裝 16
2.2 二氧化矽形成與發展 18
2.2.1 二氧化矽水解反應 18
2.2.2 二氧化矽縮合反應 19
2.2.3 影響二氧化矽顆粒大小 20
2.3 多孔性材料 21
2.3.1 中孔洞二氧化矽的重要性 22
2.3.2 中孔洞二氧化矽發展 23
2.4金奈米粒子的還原與機制 26
2.4.1 金與中孔洞二氧化矽觸媒應用 27
2.5 二氧化鈦發展 28
2.5.1 二氧化鈦中空管製備 28
2.6 光催化反應 30
2.6.1 二氧化鈦光觸媒催化原理 31
第三章 實驗 34
3.1 實驗藥品 34
3.2 實驗器材 35
3.3 合成聚胺基酸 36
3.3.1 合成起始劑 36
3.3.2 乾燥溶劑 37
3.3.3製備α-胺基酸的N-carboxyanhydrides(NCAs) 37
3.3.4 胺基酸聚合反應 38
3.3.5 切除聚胺基酸保護基R-group 39
3.4 合成無機氧化物材料 40
3.4.1 二氧化矽中空管的製備 42
3.4.2二氧化鈦中空管的製備 45
3.5 分析儀器 47
3.5.1傅立葉轉換之紅外線光譜儀 47
3.5.2場發射掃描式電子顯微鏡 48
3.5.3穿透式電子顯微鏡 49
3.5.4 X光繞射分析儀 50
3.5.4熱重分析儀 51
3.5.6紫外光/可見光譜儀 52
3.5.7反射式紫外/可見光光譜儀 53
3.5.8 膠滲透層析儀 53
3.5.9 氮氣吸脫附儀 55
第四章 結果與討論 60
4.1實驗聚胺基酸分子鏈長和樣品定義 60
4.2 二氧化矽複合材料管之形成 62
4.2.1中孔洞二氧化矽管的形態和構造 68
4.2.2對二氧化矽管壁的影響 75
4.2.3金奈米粒子/中孔洞二氧化矽管的形成 80
4.2.4金奈米粒子/中孔洞二氧化矽管觸媒應用 89
4.3二氧化鈦複合材料管之形成 94
4.3.1二氧化鈦管的物理性質和對管壁的影響 99
4.3.2二氧化鈦的結晶形成與影響 105
4.3.3二氧化鈦光觸媒應用 111
第五章 結論 118
5.1 總結與未來展望 118
文獻參考 122
自述 131



表目錄
表 2.1 從1995~2007年,利用不同模板法配合其他合成技術製備無機氧化物奈米管總整理[45] 13
表 4.1 以GPC測量出聚胺基酸高分子鏈的分子量、聚合度和PDI 61
表 4.2 BET氮氣吸脫附儀,各個樣品1~11的BET數據 71
表 4.3 從圖 4.5中,整理出11個樣品的每層平均管壁厚度 79
表 4.4 將圖 4.18的線性迴歸線,數據化所整理出的表 93
表 4.5 從SEM和TEM影像觀察,不同分子量下各溶液樣品中的每層平均管壁厚度 102
表 4.6將(圖 4.34)的線性迴歸線,取間隔24 min為一定點,量測到120 min共五點,13個樣品經過數據化和配合(表 4.5)管壁厚度所整理出的表 117



圖目錄
圖 1.1 以團聯聚胺基酸形成球狀的二氧化矽[29] 7
圖 1.2 高分子化學結構式 (a)改質氧化矽的表面接上PLL稱做t-PLL (b)PAA (c) PLGA 和 (d)PLAA[33] 9
圖 1.3 t-PLL結合其他高分子形成不同二級結構,圖由左到右分別為,螺旋結構與PAA結合、片狀與PLGA結合和任意捲曲狀與PLAA結合[33] 9
圖 2.1 從1995~2007年,利用模板法在各個領域的應用[45] 14
圖 2.2 層層自組裝以浸入方式的示意圖,上圖為巨觀吸附動作;下圖為微觀靜電吸附力作用[58] 15
圖 2.3 利用模板法製備出不同形態的LBL結構 a類為在空氣中形成 b類為再空氣和液體接觸面形成 c類為在液液接觸面形成,其中第一纇a1.微懸臂樑、a2.微管柱、a3.微膠囊、a4.平板薄膜;第二類b1.水滴狀、b2.氣體球和第三類c1.微方塊、c2.微中空球[65-67] 17
圖 2.4 為Mobil公司開發出的三種中孔洞二氧化矽的單位晶格排列,從左到右為,二維六角管柱堆積MCM-41、三維立體方塊MCM-48和二維層狀排列MCM-50[79] 24
圖 2.5 由Beck等人提出形成MCM-41可能的反應機制示意圖,途徑1.為先形成液晶相的微胞管柱,在進行六角堆積排列;途徑二為直接矽化成六角堆積二氧化矽管柱[78] 25
圖 2.6 由Beck等人提出不同孔洞單位的晶格的排列形成的反應機制,微結構由微胞在形成較複雜的管柱、層狀排列還是雙層結構,最後才形有相同序列的排列堆積[79] 25
圖 3.1 為合成無機氧化物二氧化矽和二氧化鈦的流程示意圖 41
圖 3.2凝膠滲透層析儀的分析圖譜,最上面為綠線分析粒徑、中間為藍線分析黏度和最下面的紅線分析折射率 54
圖 3.3 五種不同吸附狀況的等溫吸脫附曲線[96] 56
圖 3.4 由IUPAC定義遲滯現象的四種形態[97] 57
圖 4.1 FTIR光譜圖,測量在三個不同溶液下,未經高溫鍛燒前,合成出的二氧化矽複合管 65
圖 4.2 EDS元素分析,分析複合材料(PLL/PLGA)n/TMOS 66
圖 4.3 TGA熱重分析,以(Lys380/Glu370)9I為分析樣品,由圖可得知有機和無機部份的比例 66
圖 4.4 SEM影像,在溶液III下經過500°C鍛燒後,合成不同層數下的二氧化矽管A. (Lys210/Glu190)5 B. (Lys210/Glu190)9和C. (Lys210/Glu190)13 69
圖 4.5 TEM影像,經過500°C鍛燒後在不同分子量不同溶液但相同層數下合成出的二氧化矽中空管分別為1.(Lys120/Glu80)9III、2.(Lys145/Glu125)9III、3.(Lys210/Glu190)9III、4.(Lys340/Glu125)9III、5.(Lys340/Glu370)9III、6.(Lys145/Glu125)9II、7.(Lys210/Glu125)9II、8.(Lys340/Glu125)9II、9.Unknow1-I、10.Unknow2-I 和11. (Lys210/Glu125)9I 70
圖 4.6 BET等溫吸脫附曲線,在三個不同溶液下,各個樣品1~11 71
圖 4.7 BJH孔徑分佈圖,在三個不同溶液下,各個樣品1~11 71
圖 4.8 TEM影像,在溶液III下經過500°C鍛燒後,相同分子量和相同層數下,不同鹽離子濃度 A. 0.1M NaCl 和 B. 0.5M NaCl 77
圖 4.9 TEM影像,在溶液III下經過500°C鍛燒後,相同分子量不同層數的二氧化矽中空管A.(Lys340/Glu370)5III、B.(Lys340/Glu370)9III和C.(Lys340/Glu370)13III 78
圖 4.10 UV-vis光譜圖動態分析,將Glu125當作還原劑,在鹼性和中性金鹽溶液條件下進行500 min的還原金奈米粒子 82
圖 4.11 TEM影像,在不同pH值下由Glu125還原金奈米粒子 A.中性水溶液和B. pH11.2 水溶液 82
圖 4.12 TEM影像,(A, B)分別為在不同影像倍率下(Lys120/Glu80)9III在鹼性金鹽溶液;(C, D)分別為在不同影像倍率下(Lys140/Glu80)9III在中性金鹽溶液,進行合成金/二氧化矽管經過95°C後 84
圖 4.13 SEM影像,樣品(Lys140/Glu80)9AuIII,在經過400°C鍛燒並恆溫10 hr後 86
圖 4.14 TEM影像,在溶液III下合成金/二氧化矽管,各個不同分子量比例、層數、鍛燒溫度和鍛燒時間的樣品1.(Lys120/Glu80)9AuIII-500°C-10hr、2.(Lys140/Glu80)9AuIII-500°C-10hr、3. (Lys140/Glu80)9AuIII-400°C-3hr、4.(Lys140/Glu80)9AuIII-400°C-10hr、5.(Lys340/Glu145)5AuIII-500°C-10hr、 87
圖 4.15 UV-vis光譜圖,分析PC薄膜在鹼性金鹽溶液下,浸入1~3天,隨天數增加金粒子吸收值也跟著增加 88
圖 4.16 UV-vis光譜圖,分析對-硝基酚(p-nitrophenol)不同濃度下,做吸收度對濃度的檢量線 90
圖 4.17 UV-vis光譜圖,隨時間降解實驗,量測金/二氧化矽管觸媒隨時間降解溶液中對-硝基酚(p-nitrophenol),其吸收度隨時間趨於平緩 91
圖 4.18 利用檢量線(圖 4.16)和隨時間降解實驗(圖 4.17)的數據,可以劃出以lnC(濃度mM)對時間(s)的降解反應速率圖,以每1 mg的樣品為單位,可分別在6個樣品得到線性迴歸線,讀出k值 93
圖 4.19 FTIR光譜圖,測量在三個不同溶液下,未經高溫鍛燒前,合成出的二氧化鈦複合管 96
圖 4.20 SEM影像,在經過95°C乾燥後,樣品(Lys140/TiBALDH)10III 98
圖 4. 21 EDS元素分析,分析複合材料(PLL/TiBALDH)n 98
圖 4.22 TGA熱重分析,以(Lys140/TiBALDH)10II為分析樣品,由圖可得知有機和無機部份的比例 99
圖 4.23 SEM影像,經過鍛燒450°C後,不同溶液下得到的樣品A.(Lys140/TiBALDH)10I、B. (Lys140/TiBALDH)10II和C.(Lys140/TiBALDH)10III 102
圖 4.24 TEM影像,鍛燒450°C後,不同溶液下得到的樣品A. (Lys140/TiBALDH)10I、B. (Lys140/TiBALDH)10II、C.(Lys140/TiBALDH)10III和D. (Lys340/TiBALDH)10III 103
圖 4.25 TEM影像,鍛燒450°C後,合成(Lys210/TiBALDH)10III在不同NaCl濃度水溶液下A. 0.2M、B. 0.35M和C. 0.5M 104
圖 4.26 XRD光譜圖,經過鍛燒450°C後,在不同溶液下合成1.(Lys140/TiBALDH)10I、2. (Lys140/TiBALDH)10II和3.(Lys140/TiBALDH)10III,最底下柱狀圖為銳鈦礦相database 106
圖 4.27 XRD光譜圖,經過鍛燒950°C後,在不同溶液下合成1.(Lys140/TiBALDH)10I、2. (Lys140/TiBALDH)10II和3.(Lys140/TiBALDH)10III,最底下柱狀圖為金紅石相database 108
圖 4.28 SEM影像,經過鍛燒950°C後,不同溶液下得到的樣品A. (Lys140/TiBALDH)10I、B. (Lys140/TiBALDH)10II和C.(Lys140/TiBALDH)10III 108
圖 4.29 TEM影像,鍛燒950°C後,在溶液I下不同層層自組裝層數得到的樣品A. (Lys140/TiBALDH)5I和B. (Lys140/TiBALDH)10I 109
圖 4.30 反射式UV-vis光譜圖,經過450°C鍛燒後,分析不同溶液下二氧化鈦管 110
圖 4. 31反射式UV-vis光譜圖,經過950°C鍛燒後,分析不同溶液下二氧化鈦管 111
圖 4.32 UV-vis光譜圖,針對Reactive Black B (RBB)在不同濃度下,做吸收度對濃度的檢量線 113
圖 4.33 UV-vis光圖譜,以樣品(Lys140/TiBALDH)10在不同溶液下表示之,進行光催化反應降解RBB溶液,以每24 min為間隔表示時間對吸收度的變化 116
圖 4. 34利用檢量線(圖 4.32)和隨時間降解實驗(圖 4.33)的數據,可以劃出以lnC(濃度ppm)對時間(min)的降解反應速率圖,每0.6 ml/mg的樣品為單位,可分別在13個樣品得到線性迴歸線,讀出k值,這裡僅以樣品(Lys140/TiBALDH)10在不同溶液下表示之 116
圖 5.1 TEM影像,以途徑二取代途徑一,(PLL/TMOS)5交替疊出五層的二氧化矽管 120
圖 5.2 SEM影像,以途徑一取代途徑二,(PLL/PLGA)9/TiBALDH交替疊層出九層的二氧化鈦管 121


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系統識別號 U0026-1507201113222800
論文名稱(中文) 酸鹼敏感型聚胺基酸水膠:特性探討及仿生多孔性複合材料之合成
論文名稱(英文) pH-Sensitive Polypeptide Hydrogels: Characterization and Biomimetic Synthesis of Porous Oxides
校院名稱 成功大學
系所名稱(中) 化學工程學系碩博士班
系所名稱(英) Department of Chemical Engineering
學年度 99
學期 2
出版年 100
研究生(中文) 陳姵珊
學號 n36984161
學位類別 碩士
語文別 中文
口試日期 2011-06-15
論文頁數 113頁
口試委員 指導教授-詹正雄
口試委員-林睿哲
口試委員-陳美瑾
口試委員-胡晉嘉
關鍵字(中) 聚胺基酸
水膠
複合材料
交聯
仿生合成
二氧化矽
關鍵字(英) polypeptide
hydrogels
hybrid materials
crosslink
biomimetic synthesis
porous silica
學科別分類
中文摘要 本研究是利用聚胺基酸高分子(polypeptide)上的胺基與交聯劑(genipin)進行反應後製備出具有酸鹼敏感型的水膠,藉由改變聚胺基酸高分子的分子量、親疏水鏈段比、聚胺基酸溶液濃度以及加入交聯劑與高分子的莫耳數比進行調控和探討水膠物理性質的變化,並可利用仿生合成方法進行有機/無機複合材料的製備,最後將有機物質經由鍛燒去除可得到仿生多孔性的二氧化矽。
在第一部分的研究中,我們控制胺基酸的分子量及親疏水鏈段比再經由開環聚合後就可得到聚胺基酸高分子,接著再加入交聯劑於聚胺基酸溶液中即可製備出聚胺基酸水膠。藉由交聯時間及膨潤比測量、機械強度以及生物毒性測試、FTIR和SEM來瞭解聚胺基酸水膠的物理性質,並做進一步的探討。從交聯時間及機械強度的結果中發現聚胺基酸的分子量、親疏水鏈段比、聚胺基酸溶液濃度以及交聯劑莫耳數比皆會影響物理性質;由FTIR中可發現聚胺基酸高分子經過交聯反應後二級結構會有些許的改變;由SEM可觀察到水膠的結構是具有高孔洞性及孔洞大小約為10~30 μm;膨潤比的結果可得知聚胺基酸水膠的膨潤比會隨酸鹼值不同而有改變;材料生物毒性的測試說明聚胺基酸經過交聯反應後可提高細胞的存活率。
第二部分的研究是利用仿生合成方法進行製備聚胺基酸-二氧化矽之有機/無機複合水膠材料。由TGA結果顯示控制矽化作用時間可得到不同有機/無機含量的複合材料;從FTIR的結果中可確定有二氧化矽的形成;在機械強度測試中可知聚胺基酸-二氧化矽複合水膠材料的強度會隨著矽化作用時間增加而下降;而聚胺基酸-二氧化矽複合水膠材料經由生物毒性測試後可得知此材料對於纖維母細胞來說是不具有生物毒性,故此新穎複合材料極具有潛力可應用於蛋白質/藥物的包覆及傳輸,以及組織工程中;最後再經由鍛燒後的TEM及BET結果可得知二氧化矽是具有多孔性的結構,且此孔洞大小的主要分布是介於2~10 nm。
英文摘要 In this study, pH-sensitive polypeptide hydrogels were prepared by using genipin to crosslink amine groups on polypeptide. We investigated the influence of the molecular weight of polypeptide, ratio of hydrophilic and hydrophobic segments, polypeptide solution concentration and molar ratio of genipin and polypeptide on the properties of the as-prepared hydrogels. The polypeptide-silica organic/inorganic hybrid materials can be prepared using biomimetic synthesis approach. According to our study, the obtained silicas after calcination were porous.
In the first part, homopolypeptides and amphiphilic block copolypeptides with different molecular weights or block ratios were via the ring-opening polymerization and genipin was used to crosslink these polypeptides in aqueous solution to form hydrogels. The properties of as-prepared hydrogels were characterized by a variety of analytical techniques including gelation time and swelling ratio measurements, compression and cytotoxicity tests, FTIR, and SEM. The results show that the gelation time and compressive modulus were influenced by the molecular weight, block ratio, polypeptide concentration, and the molar ratio of genipin and polypeptide. FTIR analysis revealed that the polypeptides underwent conformational changes after crosslinking reaction. The structure of hydrogels was found to be highly porous with sizes between 10 and 30 μm by SEM characterization. The degree of swelling was found to vary with the solution conditions (that is, pH value). The cytotoxicity tests suggested that the genipin-crosslinked polypeptide hydrogels can enhance the cell viability comparing with the polypeptides without crosslinking.
The second part demonstrated that the polypeptide-silica organic/inorganic hybrid materials can be prepared using biomimetic synthesis approach. TGA analysis showed that the weight ratio of the organic and inorganic compounds in the hybrid materials can be controlled by varying the silification time. FTIR analysis revealed that the covalent bond formation between polypeptide and silica. The compressive strength of polypeptide-silica hydrogel was decreased with increasing the silification time. The as-prepared hybrid materials were found to be not cytotoxic to fibroblast cells, so these novel hybrid materials have potential applications including protein/drug delivery, encapsulation, and tissue engineering. Based on TEM and BET characterization, the silicas obtained by calcination were to found be highly porous with sizes mainly between 2 and 10 nm.
論文目次 摘要 I
Abstract III
誌謝 V
目錄 VI
圖目錄 XI
表目錄 XVII
第一章 緒論 1
1.1 前言 1
1.1.1 生物性材料簡介 1
1.1.2 水膠的發展 3
1.2 研究動機與目的 4
第二章 文獻回顧 5
2.1 胺基酸基本性質 5
2.1.1 簡介胺基酸及其結構 5
2.1.2 蛋白質的結構 8
2.2 水膠的定義與種類 12
2.2.1 水膠的定義 12
2.2.2 智慧型水膠的種類 12
2.3 水膠的物理性質 16
2.3.1 膨潤行為理論 16
2.4有機/無機複合材料 19
2.4.1 生物材料與礦化作用的發展 19
2.4.2 有機/無機複合材料簡介 20
2.4.3 聚賴胺酸沉析二氧化矽 21
2.5多孔性複合材料 26
2.5.1 中孔洞二氧化矽的重要性 27
2.5.2 中孔洞二氧化矽發展 28
第三章 實驗步驟與實驗原理 30
3.1 實驗藥品與儀器設備 30
3.1.1 實驗藥品 30
3.1.2 儀器設備 32
3.2 聚胺基酸高分子之合成 34
3.2.1 起始劑之合成 34
3.2.2 溶劑之乾燥方式 34
3.2.3 胺基酸之N-carboxyanhydrides(NCAs)的製備 35
3.2.4 聚胺基酸之開環反應 36
3.2.5 聚胺基酸之保護基(R-group)去除 37
3.3 聚胺基酸水膠之物理性質測試 38
3.3.1 聚胺基酸水膠之製備 38
3.3.2 聚胺基酸水膠之凝膠時間點(Gelation time)測試 38
3.3.3 聚胺基酸水膠之膨潤比測試 38
3.3.4 聚胺基酸水膠之機械性質 39
3.3.5 聚胺基酸水膠之生物毒性測試 39
3.4 聚胺基酸水膠之二氧化矽複合材料製備 40
3.4.1 聚胺基酸水膠之沉析無機物 40
3.4.2 聚胺基酸水膠之中孔複合材料製備 40
3.5 特性分析與性質測試 40
3.5.1 核磁共振光譜儀 40
3.5.2 凝膠滲透層析儀 41
3.5.3 高解析場發射掃描式電子顯微鏡 43
3.5.4 穿透式電子顯微鏡 44
3.5.5反射式紅外線光譜儀 45
3.5.6 紅外線光譜儀 46
3.5.7 圓二色光譜儀 47
3.5.8 萬能材料試驗機 49
3.5.9 熱重分析儀 50
3.5.10 氮氣吸脫附儀 51
第四章 結果與討論 57
4.1聚胺基酸高分子之定義 57
4.1.1聚胺基酸高分子之比例 57
4.1.2 聚胺基酸高分子之分子量與鏈長 60
4.2聚胺基酸水膠之物理性質 62
4.2.1聚胺基酸水膠之形成 62
4.2.2聚胺基酸水膠之交聯時間(Gelation time) 65
4.2.3聚胺基酸水膠之二級結構變化 69
4.2.4聚胺基酸水膠之型態與構造 75
4.2.5聚胺基酸水膠之膨潤比(Swelling ratio) 78
4.2.6聚胺基酸水膠之機械強度 81
4.2.7聚胺基酸水膠之生物毒性測試 85
4.3仿生多孔性複合材料 86
4.3.1 聚胺基酸-二氧化矽複合水膠材料之製備 86
4.3.2 聚胺基酸-二氧化矽複合水膠材料之二級結構 89
4.3.3 聚胺基酸-二氧化矽複合水膠材料之機械強度 91
4.3.4 聚胺基酸-二氧化矽複合水膠材料之生物毒性 93
4.3.5 二氧化矽複合材料之形態 94
4.3.6二氧化矽複合材料之中孔(mesopore)特性 97
第五章 結論 105
參考文獻 108
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系統識別號 U0026-2007201016261901
論文名稱(中文) 胍與吡唑衍生物合成新製程的開發
論文名稱(英文) Development of New Synthetic Methodologies for Guanidine and Pyrazole Derivatives
校院名稱 成功大學
系所名稱(中) 化學系碩博士班
系所名稱(英) Department of Chemistry
學年度 98
學期 2
出版年 99
研究生(中文) 陳俊言
學號 l3895106
學位類別 博士
語文別 中文
口試日期 2010-07-15
論文頁數 116頁
口試委員 指導教授-葉茂榮
口試委員-莊治平
口試委員-周金興
口試委員-陳銘田
口試委員-施美秀
關鍵字(中) 異硫氰酸酯
異氰酸酯
氰胺

5-胺基吡唑
關鍵字(英) isothiocyanates
isocyanates
cyanamides
guanidines
5-aminopyrazoles
學科別分類
中文摘要 胍 (guanidines) 是一種具多種用途的藥理活性分子,因此在文獻上有許多方法被建立用來合成胍。而氰胺 (cyanamides ) 則是合成胍的前驅物,且氰胺也是一種抗腫瘤 (tumor inhibitors) 藥物。合成氰胺有許多方法,然而受限於合成條件的關係,在產率或是製備上均有其缺點。本論文首先開發合成氰胺的新方法,利用商業化且具立體障礙的鹼—雙(三甲基矽基)醯胺鈉 [NaN(SiMe3)2] 化合物和異硫氰酸酯進行反應,合成中高產率且具有藥理活性的氰胺化合物,此一新合成方法可應用於各種類型的異硫氰酸酯。接著我們藉著催化劑的使用,結合上述的新方法,建立一個新的一鍋反應來合成胍化合物;分子生物活性的篩檢資料顯示,有幾個我們合成出的胍分子在抗腫瘤測試上有不錯的效果。
吡唑為主體結構的衍生物已被研究出具有廣泛生物活性及藥理作用,近年來也有不少關於其在發光材料上應用的報導;基於吡唑分子的重要性,我們針對吡唑環上的鹵素原子,利用鈀催化劑[PdCl2(PPh3)2]將其氫化,得到高產率的吡唑分子,並做了一系列催化劑與其他條件的研究。最後為利用銅(CuI)催化系統來使5-胺基吡唑(5-aminopyrazole)與芳香鹵化物(arylhalide)進行碳氮鍵的偶合反應,我們找到一個適合的反應條件,來改善5-胺基吡唑其親核性較差的缺點,並能選擇性得到中高產率的兩種偶合產物。
英文摘要 The guanidinium group is present in many natural and synthetic biologically active compounds. Due to its broad spectrum of activity, the guanidine unit has been intensively studied as a synthetic goal and a diversity of new methods has been developed. Cyanamides are important precursors in the synthesis of guanidines. They also exhibit apparent tumor growth inhibition activity. Many of reagents are developed as a cyano cation (CN+) agent to synthesize the cyanamide products. Although their syntheses are straightforward, they do not produce quantitative yields and require tedious purification procedures. Herein, we report a transformation method of isothiocyanates to cyanamides using the commercially available alkali amide NaN(SiMe3)2. Additionally, we expanded the reaction to one pot synthesis of guanidines hydrochloride by using catalyst. Screening data indicated that several compounds exhibited significant in vitro activities against numerous human tumor cell.

Pyrazoles are an important family of heterocyclic compounds due to their wide range of pharmacological proprieties. In particular, modified pyrazoles are also the basis of various active material. For this reason, we report the use of palladium chloride in the presence of triphenylphosphine to remove the halogen atom in pyrazoles. The reaction went rapidly and efficiently. Finally, we have developed a new and efficient copper-catalyzed C-N bond formation for 5-aminopyrazoles with arylhalides. The reaction conditions for the copper-mediated N-arylation of heteroarylamines were optimized. The desired compounds of N-monoarylation or N-diarylation of 5-aminopyrazoles were synthesized in different reaction conditions.

論文目次 第一篇 異硫氰酸酯與異氰酸酯轉換成胍鹽酸鹽及其在抗癌細胞上的應用

第一章 以矽氮化合物將異硫氰酸酯轉換成氰胺
第一節 緒論…………………………………………………1
第二節 結果與討論…………………………………………5
第三節 結論…………………………………………………10
第四節 實驗部分……………………………………………11
第五節 參考文獻 …………………………………………16

第二章 一鍋反應中將異硫氰酸酯與異氰酸酯轉換成胍鹽酸鹽之研究
第一節 緒論…………………………………………………19
第二節 結果與討論…………………………………………27
第三節 結論…………………………………………………39
第四節 實驗部分……………………………………………40
第五節 參考文獻……………………………………………49

第二篇 吡唑衍生物的合成
第一章 1,3-雙取代-5-鹵素吡唑的去鹵素反應
第一節 緒論…………………………………………………52
第二節 結果與討論…………………………………………56
第三節 結論…………………………………………………64
第四節 實驗部分……………………………………………65
第五節 參考文獻……………………………………………75

第二章 5-胺基吡唑(5-aminopyrazole)與芳香鹵化物(arylhalide)間的碳氮鍵偶合反應
第一節 緒論…………………………………………………80
第二節 結果與討論…………………………………………85
第三節 結論…………………………………………………96
第四節 實驗部分……………………………………………98
第五節 參考文獻 …………………………………………115
參考文獻 第一篇
第一章
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------------------------------------------------------------------------ 第 14 筆 ---------------------------------------------------------------------
系統識別號 U0026-2008201319593900
論文名稱(中文) 離胺基氧化酶透過膠原蛋白/整合素相關聯的訊號對細胞硬性及移動的調控
論文名稱(英文) Lysyl oxidase modulates cell stiffness and migration through collagen/integrin-associated signaling
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 101
學期 2
出版年 102
研究生(中文) 呂瑜珍
學號 S46004103
學位類別 碩士
語文別 英文
口試日期 2013-07-26
論文頁數 52頁
口試委員 指導教授-楊倍昌
口試委員-蔡曜聲
口試委員-王仰高
關鍵字(中) 離胺基氧化酶
細胞移動
細胞硬度
關鍵字(英) lysyl oxidase
cell migration
cell stiffness
學科別分類
中文摘要 細胞周圍的微環境主要是由各種間質細胞和胞外基質所組成,他們在調控細胞上扮演重要角色。過去的研究發現腫瘤的微環境與一般組織有很大的不同,例如過多的間質細胞和胞外基質累積,甚至比一般組織來的硬;而這些改變都能促進癌細胞的存活與轉移。離胺基氧化酶(lysyl oxidase, LOX)為一種胞外基質的調控者,藉由氧化膠原蛋白與彈性纖維,促使他們產生交聯(cross-linking)反應,以維持胞外基質的硬性及結構的穩定性。在許多腫瘤中發現離胺基氧化酶有過度表現的情況;文獻中也指出離胺基氧化酶所導致的過多膠原蛋白交聯會使膠原蛋白的受體-整合素(integrin)過度活化,進而影響下游蛋白的表現,促進癌細胞的惡化。為進一步探討離胺基氧化酶在癌化細胞中所扮演的角色,在本篇研究中,我們建立了離胺基氧化酶過度表現的腦癌細胞株 (U118-MG)。實驗發現離胺基氧化酶能夠促進U118-MG單細胞遷移及傷口癒合的能力;同時過度表現離胺基氧化酶U118-MG的培養液 (LOX/CM) 也能有效提高MCF7及HepG2的傷口癒合的速度。且這些現象在加入了β-amino-propionitrile (離胺基氧化酶抑制劑)後,就能夠抑制。原子力學顯微鏡測試也發現過度表現離胺基氧化酶U118-MG及經過LOX/CM處理的細胞都具有較高的細胞硬度;但利用攜帶膠原蛋白靜默RNA之慢病毒轉染降低U118-MG的膠原蛋白表現後,就能抑制離胺基氧化酶的作用。這些結果顯示膠原蛋白的交聯在細胞硬度上扮演一個重要的角色。我們近一步發現利用抗體使整合素失去作用後,也能有效阻止離胺基氧化酶所導致的細胞硬度上升現象。離胺基氧化酶也會去促進整合素下游蛋白的磷酸化,例如FAK、SRC、ERK;相同的,此種活化情形在抑制膠原蛋白及整合素的情況下就不會發生。綜合以上的結果顯示離胺基氧化酶可能藉由調控膠原蛋白/整合素相關的訊息路徑去促進細胞的移動及硬度。我們在未來會近一步去探討離胺基氧化酶對基質修飾後所導致的整合素訊息傳遞在腫瘤生長上的影響。
英文摘要 The microenvironment surrounding the cancer cells is composed of stromal cells and a variety of extracellular matrix (ECM) proteins. Increases in stromal cells and ECM deposition promote tumor survive and invasion. Besides, tumor nodules are stiffer than normal tissues. Lysyl oxidase (LOX) is a copper-dependent enzyme, and catalyzes the cross linkage of collagen and elastin, which contributes to matrix stiffness. Crosslinking of collagen results in integrin activation and new proteins expression, which are positively correlated with tumor malignancy. Elevated expression of LOX has been detected in many types of tumors. To study the molecular mechanism underlying the tumor promoting activity of LOX, LOX gene has been ectopically expressed in U118-MG (human glioma cells). LOX-overexpression significantly increased single cell migration and wound healing ability. Besides, 48-h conditioned media from LOX-overexpression U118-MG cells (LOX/CM) also accelerated the polarized migration of MCF7 and HepG2. Treating cells with β-amino-propionitrile, an irreversible LOX antagonist, effectively diminished this augmented cell motility in tumor cells tested. A higher level of cell stiffness was observed in LOX-overexpression cells and LOX/CM-treated cells detected by atomic force microscopy as compared with those of their respective control groups. Down regulation of collagen IV by lentiviral-mediated transduction inhibited the LOX-induced stiffness elevation of U118-MG indicating that collage crosslinking played a role in cell stiffness. Blockage of integrin, the major receptor of collagen, averted the LOX-associated change of stiffness in U118-MG, MCF7, and HepG2. In addition, LOX-overexpression and LOX/CM-treatment induced the activation of several downstream kinases of integrin signaling pathway, such as FAK (Y397, Y861), Src (Tyr416), and Erk, but the induction was not found in collagen-deficient U118-MG and anti-1-treated cells. In summary, our study suggested that LOX increased the cell migration and stiffness by stimulating the collagen/integrin signal pathway. In the future, we will analyze the contribution of integrin signal transduction initiated by LOX-dependent ECM crosslinking to tumorigenesis.



論文目次 考試合格證明.........................................I

中文摘要............................................II

Abstract...........................................III

Acknowledgement....................................V

Contents...........................................VII

Figure index.......................................IX

Introduction.......................................1

Tumor-associated microenvironment..................1

Alteration of ECM in tumor progression.............1

The role of lysyl oxidase (LOX) in cell stiffness and migration..............2

Rationale of this study............................3

Materials and Methods..............................4

Materials..........................................4

Media and Buffer preparation.......................8

Methods............................................13

Mini plasmid isolation.............................13

Transformation.....................................14

Cell culture.......................................15

Transfection.......................................15

Reverse-transcription polymerase chain reaction (RT-PCR)............16

Western blot.......................................17

Cell proliferation assay...........................18

Single cell motility assay.........................19

In vitro wound healing assay.......................19

Stiffness analysis.................................19

LOX activity.......................................20

Results ...........................................21

Overexpression of LOX gene in human glioma cells............21

Overexpression of LOX gene increased the cell motility of U118-MG cells............21

Conditioned medium of U118-LOX (LOX/CM) enhanced cell migration of various cancer cells............22

LOX increased cells stiffness through collagen/integrin-associated pathway............23

LOX induced the phosphorylation of FAK, SRC and ERK...................24

Discussion.....................26

LOX in cancer cells migration.......................26

The role of LOX in cell stiffness...................27

References..........................................29

Figures and figure legends..........................33


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系統識別號 U0026-2108201216263500
論文名稱(中文) 17-丙烯胺基-17-去甲氧基格爾德黴素對抗腸病毒七十一型之研究
論文名稱(英文) The effect of 17-allylamino-17-demethoxygeldanamycin on enterovirus 71 replication in vitro and in vivo
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 100
學期 2
出版年 101
研究生(中文) 吳思瑩
學號 S46994023
學位類別 碩士
語文別 中文
口試日期 2012-07-24
論文頁數 61頁
口試委員 指導教授-余俊強
口試委員-陳舜華
口試委員-王舜德
關鍵字(中) 腸病毒七十一型
17-丙烯胺基-17-去甲氧基格爾德黴素
熱休克蛋白九十
關鍵字(英) EV71
17-AAG
Hsp90
學科別分類
中文摘要 腸病毒七十一型是屬於微小病毒科的腸病毒屬,嬰幼兒童感染會發生手口足症,偶爾病發重症,例如腦膜腦炎、肺水腫、或是神經性疾病,更嚴重者會致死。現今在臨床上針對腸病毒七十一型感染的用藥僅限於米力農(Milrinone)、靜脈注射免疫球蛋白(IVIG)及一些支持性療法,因此希冀可以尋找更多其他有效治療用藥。熱休克蛋白九十(Heat shock protein 90;Hsp90)是宿主細胞內重要的伴隨蛋白,可以幫助胞內蛋白正確摺疊,執行功能。由於病毒是絕對寄生,可以利用宿主蛋白幫助自己複製,微小病毒科若干病毒已證實會利用Hsp90協助病毒蛋白成熟,而且其複製可被17-丙烯胺基-17-去甲氧基格爾德黴素 (17- allylamino- 17- demethoxygeldanamycin ;17-AAG)抑制,因此我們要探討是否可用17-AAG抑制腸病毒七十一型的感染。首先,利用噻唑藍溴化四唑方法和臺盼藍細胞存活率分析法確認17-AAG在1 µM的濃度以下,於24小時內不會對細胞產生即毒性。而後在溶斑檢定法及半數組織培養感染劑量確定可以有效抑制腸病毒七十一型的複製,並且可以減少病毒所造成的細胞病變情形。相關實驗也證實,在細胞感染腸病毒七十一型後,越早給予17-AAG處理,抑制病毒的效果越好。在動物實驗部分,我們證實感染腸病毒七十一型的三日齡仔鼠若給予17-AAG治療,會有較高的存活率。總的來說,在體內及體外試驗都證明17-AAG可以抑制腸病毒七十一型的複製及感染,可以提供腸病毒七十一型臨床用藥的一個選擇。
英文摘要 Enterovirus 71 (EV71) belongs to the Picornaviridae family. EV71 infection may cause hand, foot and mouth diseases (HFMD) in infants and young children, but sometimes result in severe complications, including acute encephalitis and some neurological diseases, even death. Nowadays, the clinical treatments are limited to Milrinone and supporting treatment. We hope to find a new treatment for EV71 infection. Heat shock protein 90 (HSP90) is a major chaperon in host cells. It often helps cellular protein fold correctly. Because viruses are intracellular obligate parasites, it may utilize host proteins for their replication. We hypothesized that EV71 may hijack HSP90 for its replication, and that we can use HSP90 inhibitor, 17-allyamino-17-demethoxygeldanamycin (17-AAG), to inhibit EV71 replication. We first checked the cytotoxicity of 17-AAG with MTT assay and trypan blue exclusion assay. We found that 17-AAG didn’t have acute cytotoxicity in 24 hrs at a dose lower than 1 µM. And then, we wanted to know whether 17-AAG could inhibit EV71 viral replication in vitro. RD cells were infected with EV71/4643, and 17-AAG was given in 1, 2, and 6 hrs. Viral titer of EV71 was decreased after treating 17-AAG. Finally, we wanted to test the effect of 17-AAG in vivo. The EV71/MP4-y5 infected ICR mice that treated with 2.5 or 5 mg/kg 17-AAG had higher survival rate than the DMSO control mice. In Sum, 17-AAG could inhibit EV71 replication in vitro and in vivo.
論文目次 中文摘要.................................................Ⅰ
英文摘要.................................................Ⅱ
致謝.....................................................Ⅲ
目錄.....................................................Ⅳ
圖目錄...................................................Ⅷ
第一章 緒論
一、腸病毒七十一型之病毒學簡介..............................1
1.分類.................................................1
2.基因型與結構..........................................1
3.生命週期與複製........................................1
二、腸病毒七十一型之致病機轉................................3
三、腸病毒七十一型之疾病與流行病學...........................3
四、腸病毒七十一型之預防與治療..............................4
1.抑制病毒的附著、進入與脫殼..............................4
2.抑制病毒蛋白的合成 ....................................5
3.抑制病毒蛋白3C蛋白酶 ..................................5
4.抑制病毒蛋白 2C ...................................5
5.抑制病毒蛋白 3A ...................................6
6.抑制病毒蛋白 3D RNA polymerase .......................6
7.核苷酸類似物..........................................6
五、宿主蛋白與病毒之交互作用................................7
1.轉錄起始因子..........................................7
2.HnRNP A1和K .........................................7
3.FBP1和2 .............................................8
4.Cleavage stimulation factor (CstF) -64..............8
六、伴隨蛋白..............................................8
1.伴隨蛋白的簡介........................................8
2.Hsp90與神經退化性疾病.................................10
3.Hsp90與癌症 .........................................10
4.Hsp90與病毒感染症疾病 ................................10
七、17-丙烯胺基-17-去甲氧基格爾德黴素(17-Allylamino-17-demethoxygeldanamycin,17-AAG) 之簡介....................11
第二章 研究動機與特異目標..................................12
第三章 材料與方法
一、材料.................................................14
二、方法
1.細胞培養.............................................19
2.病毒培養.............................................20
3.病毒定量:病毒斑分析法.................................21
4.病毒株鑑定:間接免疫螢光染色法..........................21
5.細胞毒性試驗..........................................22
6.病毒抑制能力試驗......................................22
7.半數組織培養感染量 (TCID50)............................23
8.17-AAG對小鼠產生的藥物毒性測試.........................23
9.測試EV71/MP4-y5的80%致死率............................23
10.腸病毒七十一型小鼠模式中,17-AAG對病毒抑制能力試驗.......23
11.西方墨點法..........................................24
12.統計分析............................................26
第四章 結果
一、腸病毒71型之病毒株鑑定.................................27
二、17-AAG之細胞毒性測試..................................27
三、17-AAG對於腸病毒七十一型在體外實驗之抑制作用.............28
四、17-AAG對於腸病毒71型在小鼠體內實驗之抑制作用.............29
五、17-AAG合併使用Ribavirin對於腸病毒七十一型之抑制作用......30
六、腸病毒七十一型與熱休克蛋白九十..........................33
第五章 討論..............................................34
參考文獻.................................................39
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系統識別號 U0026-2208201217310600
論文名稱(中文) 精胺基琥珀酸裂解酶在癌症中所扮演之角色
論文名稱(英文) The Role of Argininosuccinate Lyase in Cancer
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 100
學期 2
出版年 101
研究生(中文) 黃浩綸
學號 s58941549
學位類別 博士
語文別 英文
口試日期 2012-07-23
論文頁數 116頁
口試委員 指導教授-賴明德
召集委員-呂增宏
口試委員-沈孟儒
口試委員-馬明琪
口試委員-洪文俊
關鍵字(中) 內質網壓力
精胺基琥珀酸裂解酶
細胞週期蛋白A2
一氧化氮
關鍵字(英) ER stress
Argininosuccinate lyase
Cyclin A2
Nitric oxide
學科別分類
中文摘要 最近,在人類癌症的氨基酸代謝也被認為是不同於正常細胞的。內質網是
負責合成蛋白質和檢測環境養分之胞器。腫瘤細胞通常生長在一個不良的環
境,如營養缺乏。因此,內質網壓力反應往往在人類癌症中被觀察到。精氨酸
和天冬酰胺分別被認為是某些類型的肝癌和白血病所必需的氨基酸。胺基酸代
謝及內質網壓力之間的訊息連結有很大的程度是未知的。此外,由於精氨酸的
一些代謝產物對於細胞生長來說是重要的而且內質網壓力的誘發者包含了B 型
肝炎病毒表面蛋白及C 型肝炎病毒核心蛋白在肝癌發展過程中扮演著重要角
色。再加上C 型肝炎病毒核心蛋白被報導會誘發精胺基琥珀酸裂解酶的表現。
因此,我們測試了精胺基琥珀酸裂解酶在癌症的進展中是很重要的此一假設。
首先,我們觀察到肝癌細胞HuH-7 中表現B 型肝炎病毒的PreS2 蛋白可以誘發精胺基琥珀酸裂解酶的表現。我們也發現不論是在肝癌、乳癌及大腸直腸癌細胞株和小鼠活體肝臟中其精胺基琥珀酸裂解酶的表現均可被內質網壓力所誘
導。在檢驗臨床檢體中精胺基琥珀酸裂解酶的表現量,我們也觀察到肝癌、乳
癌及大腸直腸癌檢體中精胺基琥珀酸裂解酶過度表現的情形。從shASL 的穩定轉染細胞株中發現到當細胞中精胺基琥珀酸裂解酶的表現被壓抑時,則會抑制了細胞的生長速率和非固著依賴性生長。此外,在BALB/c 及 NOD/SCID 小鼠皮下注射腫瘤細胞的模式中,當精胺基琥珀酸裂解酶的表現被壓抑時,亦會使得其腫瘤生長變慢而延長其存活率。再者,給予腫瘤老鼠注射帶有精胺基琥珀酸裂解酶shRNA 的慢病毒也可以抑制腫瘤生長。而檢驗細胞週期相關蛋白表現時,只發現到cyclin A2 蛋白的表現量降低且其信使核醣核酸的表現量則不變。另外處理過蛋白酶抑制劑的細胞其cyclin A2 蛋白表現量降低的情形則可被回復。另一方面細胞週期的分析也顯示G2/M 週期的延遲。此外,當過度表現精胺基琥珀酸裂解酶時也可增加cyclin A2 蛋白表現量。當精胺基琥珀酸裂解酶的表現被壓抑時,細胞也有自噬作用的產生,同時細胞內的一氧化氮含量也有減少的現象,但是細胞內的精胺酸含量則不受影響。當給予細胞多量的精胺酸則並不會使細胞生長及細胞內一氧化氮含量有回復的情形。最後給予細胞額外的一氧化氮則可以部分地回復精胺基琥珀酸裂解酶表現被壓抑時所導致的細胞生長抑制之情形。過度表達cyclin A2 或是精胺基琥珀酸裂解酶則能回復精胺基琥珀酸裂解酶表現被壓抑時所產生的細胞生長抑制之情形。總而言之,此研究有以下的一些結果: (1) 內質網壓力是透過轉錄的方式來誘發精胺基琥珀酸裂解酶,(2) 我們也觀察到肝癌、乳癌及大腸直腸癌檢體中精胺基琥珀酸裂解酶有過度表現的情形,(3) 當精胺基琥珀酸裂解酶的表現被壓抑時會誘發細胞的自噬作用但會使細胞中的cyclin A2 蛋白表現量和一氧化氮含量減少,而這些現象則在抑制腫瘤生長中扮演著一定的角色。
英文摘要 Recently, the amino acid metabolism in human cancer was found to be different from that in normal cells. The endoplasmic reticulum is an organelle responsible for synthesizing proteins and sensing environmental nutrients. Cancer cells often grow in a poor environment such as nutrient deprivation. Therefore, the endoplasmic reticulum (ER) stress is often observed in human cancer. Arginine and asparagine are considered essential amino acids for certain types of liver cancer and leukemia, respectively. Until now, the signal link between endoplasmic reticulum stress and amino acid metabolism is largely unknown. Since arginine metabolic products play an important role in cell growth, and ER stress inducers including surface protein of hepatitis B virus (HBV) and core protein of hepatitis C virus (HCV) are important in the formation of hepatocellular carcinoma (HCC). We tested the hypothesis that argininosuccinate lyase (ASL) is important for the tumorigenesis. Here we first found that HBV Pre S2 could induce ASL expression in HuH-7 cells. The expression of ASL was induced by ER stress not only in liver, breast and colon cancer cells but also in mice livers. Examination of ASL expression in cancer specimens indicated that ASL was over-expressed in HCC, breast and colon cancers. Cells expressing shASL inhibited proliferation rates and anchorage -independent growth in vitro and tumor formation in vivo in BALB/c and NOD/SCID mice. Furthermore, lentiviral infection of ASL shRNA inhibited HuH-7 tumor growth in a therapeutic animal model. Examination of the expression of protein related to cell cycle revealed that only cyclin A2 was decreased. The cyclin A2 mRNA was unaltered by ASL shRNA and the proteasome inhibitor restored the expression of cyclin A2. On the other hand, analyses of the cell cycle effects of ASL shRNA revealed a G2/M delay. In addition, overexpression of ASL in liver cancer cells up-regulates cyclin A2. Autophagy was observed in the cells treated with ASL shRNA. Stable knockdown of ASL reduced NO content but did not affect arginine level. Addition of extra arginine did not increase the level of NO and cell growth. Finally, incubation with nitric oxide donor partially recovered the growth inhibition by ASL shRNA. Ectopic expression of cyclin A2 or ASL could rescue the growth attenuation in ASL-knockdown cells. In summary, our report has the following findings; (1) ER stress induced ASL transcriptionally, (2) ASL is over-expressed in HCC, breast and colon cancers, (3) knockdown of ASL caused the increase of autophagy, the decrease of cyclin A2, and the cellular NO content which is in part mediated attenuation of tumor growth.
論文目次 Page number list
Abstract in Chinese
Abstract
I. Introduction………………………………………………………………….....01
I-1. Endoplasmic Reticulum Stress……………………………………………01
I-2. Endoplasmic Reticulum Stress and cell metabolism…………………….03
I-3. Endoplasmic Reticulum Stress and Cancer………………………...........05
I-4. Cancer and Cell Metabolism………………………………………...........08
I-5. Amino Acid Metabolism in Cancer………………………………………11
I-6. Endoplasmic Reticulum Stress, Amino Acid Metabolism, and
Cancer…………………………………………………………………......14
I-7. Arginine and Argininosuccinate lyase........................................................15
I-8. Autophagy.....................................................................................................17
II. Materials and Methods…………………………………………………...........22
II-1. Cell Culture……………………………………………………………….22
II-2. Chemicals, Reagents and Antibodies……………………………............22
II-3. Reverse transcription-Polymerase Chain Reaction (RT-PCR)
Analysis…………………………………………………………….....23
II-4. Lysate collection and Western blot analysis…………………………….24
II-5. In vivo induction of ER stress by tunicamycin………………….............25
II-6. Plasmid Transfection and Reporter Gene Assay……………………….26
II-7 Tissue Samples…………………………………………………………….26
II-8. RNA interference…………………………………………………............27
II-9. Proliferation assay (MTT assay)………………………………………...28
II-10. Foci formation assay…………………………………………….............28
II-11. Soft Agar Assay………………………………………………………….28
II-12. Production and titration of lenti-virus…………………………………29
II-13. Animals and Tumor model………………………………………...........29
II-14. Treatment of HuH-7 tumor in NOD/SCID mice………………………30
II-15. Monodansylcadaverine (MDC) staining……………………….............30
II-16. Measurement of intracellular arginine content……………………….31
II-17. Intracellular nitric oxide detection……………………………………..32
II-18. Statistical analysis……………………………………………………….32
III. Results………………………………………………………………………....33
III-1. ASL expression is induced by ER stress……………………………….33
III-2. Induction of ASL by ER stress is through transcriptional
regulation………………………………………………………………...34
III-3. ASL is over-expressed in liver, breast and, colon cancer……………...34
III-4. Knockdown of ASL expression by ASL shRNA inhibited cell
growth, migration and anchorage-independent ability……………….35
III-5. Decreased tumorigenicity of shASL stable transfectants in
BALB/c and NOD/SCID mouse tumor model…………………............36
III-6. Knockdown of ASL in liver, breast and colorectal cancer cells
reduced cyclin A2 expression……………………………………..........38
III-7. Over-expression of ASL in liver cancer cells up-regulates
cyclin A2………………………………………………………………….39
III-8. Induction of cellular autophagy were observed in different kinds
of cancer cells bearing ASL shRNA…………………………………….40
III-9. Reduction of ASL expression affects cellular nitric oxide level………41
III-10. Reduced NO content of cancer cells inhibited cell growth……..........42
III-11. The cyclin A2 expression was not affected under treatment of
NOS Inhibitor ….………………………………………………………43
III-12. The NO content in cells could affect the level of cellular
autophagy………………………………………………………………44
III-13. Excess arginine did not restore either the cell growth or the
content of NO……………………………………………………..........44
III-14. Downregulation of cyclin A2 by shASL may be independent of
the activity of ASL..................................................................................45
III-15. Ectopic expression of cyclin A2 restored the inhibition of cell
growth in ASL-knockdown cells……………………………………...45
IV. Discussion...........................................................................................................47
V. Conclusions..........................................................................................................53
VI. References..........................................................................................................54
VII. Figures..............................................................................................................67
VIII. C.V.................................................................................................................116









Figure list
Fig. 1 Urea cycle and citrulline-NO cycle………………………………..............67
Fig. 2 ASL was induced in HuH-7 overexpressed HBV wild type or pre-S2
deletion mutant large surface protein……………………………….........68
Fig. 3 Induction of ASL mRNA in liver cancer cells under ER stress…………69
Fig. 4 Induction of ASL protein in liver cancer cells under ER stress…............70
Fig. 5 Induction of ASL protein in response to ER stress in vivo………............71
Fig. 6 Induction of ASL in breast cancer cells under ER stress………..............72
Fig. 7 Induction of ASL protein in colon cancer cells under ER stress…..........73
Fig. 8 ASL promoter activity was induced by TM treatment and induction
of ASL by ER stress was attenuated by actinomycin D treatment...........74
Fig. 9 Induction of ASL by ER stress was attenuated by 2-AP…………...........75
Fig. 10 Overexpression of ASL was observed in hepatocellular
carcinoma…………………………………………………………...............76
Fig. 11 Over-expression of ASL was observed in breast cancer………………..77
Fig. 12 Over-expression of ASL was observed in colon cancer………………...78
Fig. 13 Reduction of Asl expression with Asl shRNA in liver
cancer cells………………………………………………………………...79
Fig. 14 ASL shRNA inhibited cell growth in liver cancer cells…………............80
Fig. 15 ASL shRNA inhibited anchorage-independent growth growth………..81
Fig. 16 Reduction of ASL expression inhibited cell growth anchorage
-independent ability in human breast and colon cancer cells………….82
Fig. 17 ASL shRNA inhibited cell migration in ML-1 cells…………………….83
Fig. 18 shASL inhibited tumor formation (liver) in BALB/c mice……………..84
Fig. 19 shASL inhibits tumor formation (liver) in NOD/SCID mice……..........85
Fig. 20 shASL inhibits tumor formation (breast and colon) in
NOD/SCID mice……………………………………………………..........86
Fig. 21 Lenti-viral shASL inhibited tumor formation in mice…………………87
Fig. 22 ASL knockdown reduced cyclin A2 expression in different
cancer cells…………………………………………………………...........88
Fig. 23 Downregulation of ASL induces a G2/M delay…………………………89
Fig. 24 ASL knockdown does not reduce the mRNA expression of
cyclins………………………………………………………………...........90
Fig. 25 Proteasome inhibitor restores the expression of Cyclin A2……………91
Fig. 26 Over-expression of ASL upregulates cyclin A2…………………………92
Fig. 27 Over-expression of ASL upregulates cyclin A2 in protein level but
not in mRNA level…………………………………………………...........93
Fig. 28 Downregulation of ASL induces cellular autophagy in different
cancer cell lines……………………………………………………………94
Fig. 29 Downregulation of ASL induces cellular autophagy in cancer
cells…………………………………………………………………...........95
Fig. 30 The effects of autophagic inhibitors on cell growth……………………96
Fig. 31 The effects of downregulation of ASL on cellular nitric oxide levels
in liver, breast, and colon cancer cell lines……………………………...97
Fig. 32 The effects of NO inhbitor on cellular nitric oxide levels in liver,
breast, and colon cancer cell lines……………………………………….98
Fig. 33 The effects of downregulation of ASL on cellular arginine
levels……………………………………………………………………….99
Fig. 34 Inhibition of NO production decreased cell growth……………..........100
Fig. 35 Inhibition of NO production decreased cell growth
(MTT assay)………………………………………………………...........101
Fig. 36 The effects of NO donor on cell growth………………………………..102
Fig. 37 The effects of NO inhibitor on cellular cyclin A2 levels………………103
Fig. 38 The effects of NO donor on cellular cyclin A2 levels………………….104
Fig. 39 The NO content could affect the level of cellular autophagy…………105
Fig. 40 The addition of excess arginine did not affect the colony forming
ability……………………………………………………………..............106
Fig. 41 The addition of excess arginine did not affect the nitric oxide
level……………………………………………………………….............107
Fig. 42 Both wild-type and mutant-type of ASL increased cyclin A2
level……………………………………………………….........................108
Fig. 43 Ectopic expression of cyclin A2 or ASL rescued the inhibition of
cell growth in shASL stable transfectants…………………………109, 110
Fig. 44 The proposed model of ASL function in our study……………............111
Supplementary Fig. 1 ATF4 candidate binding site in the promoter region
of ASL gene…………………………………….................112
Supplementary Fig. 2 The prognosis of patients with breast cancer from the Kaplan-Meier plotter database
(ASL expression level)…………………………………….113
Supplementary Fig. 3 The prognosis of patients with breast cancer from
the Kaplan-Meier plotter database (ASL in ER
negative or ER negative and HER2 overexpression
group)……………………………………………………...114
Supplementary Fig. 4 The expression of cyclins in cells incubated in the
arginine depleted medium……………………………..115
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系統識別號 U0026-2408201323222000
論文名稱(中文) 探討腸病毒71型2C及3C蛋白基因變異之影響
論文名稱(英文) Effect of genetic variations in 2C and 3C proteins on enterovirus 71
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 101
學期 2
出版年 102
研究生(中文) 鄭惠儷
學號 t36001015
學位類別 碩士
語文別 中文
口試日期 2013-07-08
論文頁數 144頁
口試委員 指導教授-王貞仁
口試委員-林貴香
口試委員-劉校生
口試委員-張權發
關鍵字(中) 腸病毒71型
胺基酸
重組病毒
一氧化氮
關鍵字(英) Enterovirus 71
amino acid
reverse genetics viruse
nitric oxide
學科別分類
中文摘要 腸病毒71型在分類上屬於小RNA病毒科,具有一條正股的RNA基因,可以被轉譯出一條含有2,193個胺基酸的聚合蛋白。一旦病毒感染宿主細胞後,這條前驅蛋白質就會被切割成4個結構蛋白(VP1、VP2、VP3 與VP4)及7個非結構蛋白(2A、2B、2C、3A、3B、3C與3D)。自從1969年,在美國加州腸病毒71型之基因型A第一次被分離出來後,世界各地陸續都有幾波疫情爆發。根據腸病毒71型流行病學的研究可知,在1997年之後,基因型B3-B5以及C2-C5都曾在亞太地區造成嚴重的流行。而在1998年,基因型C2之腸病毒71型造成台灣第一次的大規模流行,在405例重症病例中有78名小朋友死亡。此外,在2008年與2012年則是基因型B5的病毒株造成規模不小之手足口症的流行。因此,本研究之目的是分析1998年、2008年與2012年來自輕症或重症之臨床病毒株的全長序列,希望藉此找出與疾病嚴重程度有關聯之遺傳變異位點。我們首先進行2008年或2012年之輕重症的比較,在輕症與重症間都沒有發現具有顯著差異的位點;然而將2008年與2012年全長序列作比較,則發現2012病毒株在非結構蛋白區有許多胺基酸改變之位點,這可能與B5病毒株繼2008年之後,又再次於2012年爆發有關。1998年,我們總共收集了9例死亡病例與15例非死亡病例,產生感染性克隆後再送定序,結果發現在2C的第302個胺基酸與3C的第55個胺基酸在死亡病例與非死亡病例中有所差異。在死亡病例病毒分離株的序列中,2C 302的位點傾向為白胺酸(L)多於纈胺酸(V),且3C 55的位點傾向是纈胺酸(V)多於異白胺酸(I);在非致死病例中這兩點的胺基酸出現頻率則和致死病例相反。而死亡病例中,白胺酸(L)和纈胺酸(V)同時出現的比率有56%;在非死亡病例中只有27%。為了瞭解2C 302與3C 55之位點在功能上所扮演的角色,我們利用感染性克隆之定點突變的方式,產生具有2C 302或3C 55之單點與雙點突變的重組病毒(2CL302V、3CV55I、2CL302V3CV55I),並觀察病毒突變前後的生物特性。結果顯示突變前後病毒的溫度感受性與使細胞凋亡的能力沒有太大差異;但在生長速度方面,2CL3023CV55病毒株在高MOI的情況下,在RD 與SK-N-SH兩種細胞中複製的速度都比具有2CV302、3CI55、2CV3023CI55病毒株的病毒快,而且2CL3023CV55也會產生較大型的病毒溶斑。此外,先前有許多研究已經證實,一氧化氮可抑制多種病毒與細菌的生長,包括腸病毒71型;我們的實驗也看到腸病毒71型被一氧化氮抑制的情況,且2C 302及3C 55之位點突變會改變對一氧化氮之感受性。綜合以上論述,我們認為2C 302與3C 55之位點在腸病毒71型複製的不同階段扮演不同的角色。
英文摘要 Enterovirus 71 (EV71) is a non-enveloped, positive-strand RNA virus of the Picornaviridae family. The viral genome encodes a single polyprotein precursor of 2,193 amino acids. Upon infection, this protein precursor is processed into four structural (VP1, VP2, VP3 and VP4) and seven nonstructural (2A, 2B, 2C, 3A, 3B, 3C and 3D) proteins. Several outbreaks were reported worldwide since it had been isolated firstly in California in 1969 known as EV71 genotype A. Studies on EV71 epidemiology showed that genotype B3-B5 and C2-C5 have caused large outbreaks in the Asia-Pacific region since 1997. During 1998, EV71 genotype C2 burst out the biggest outbreak in Taiwan, resulting in 405 cases of severe neurological sequelae and 78 deaths. In addition, in 2008 and 2012, genotype B5 has caused serious HFMD epidemics in Taiwan. In this study, we aim to find genetic variations between EV71 isolates in 1998, 2008 and 2012 by analyzing EV71 full-length sequences. First of all, we found no conserve difference among severe and mild cases in 2008 or 2012. Interestingly, there were several different amino acids in non-structure proteins between 2008 and 2012. These differences in non-structure proteins may correlate with the re-emergence of EV71 genotype B5 in 2012. In 1998, the 9 fatal and 15 non-fatal cases were used to produce full length clone and sequence. Two major nucleotide differences among fatal and non-fatal cases were identified at the position 4985 in 2C302 and position 5555 in 3C55. In fatal cases, leucine was identified more than valine at 2C302 and more valine than isoleucine was found at 3C55. In non-fatal cases, the frequency of amino acid expressing is opposite. The percentage of leucine and valine which expressed simultaneously in 2C302 and 3C55 is 56% among fatal cases and only 27% among non-fatal cases. In order to examine the functional role of 2C302 and 3C55, we introduced the 2C-L302V and 3C-V55I mutations, single or double, by site-directed mutagenesis to the EV71 full length infectious cDNA clone. Reverse genetics viruses (rg viruses) obtained were used to examine viral properties. The four rg viruses all showed temperature sensitive phenotype and induced similar effect of apoptosis. However, the rg virus 6359, which contained 2C-L302 / 3C-V55 showed faster growth than other mutant viruses on one step viral growth curve at high moi in both RD and SK-N-SH cells. In addition, the 6359 rg virus formed larger plaques compared to that of mutant rg viruses. In addition, it has been demonstrated that nitric oxide (NO) could inhibit the replication of many pathogens, including EV71. We found the 2C302 and 3C55 can affect the sensitivity of inhibition by NO. This study suggests amino acids at 2C302 and 3C55 play various roles in EV71 replication.
論文目次 目錄
第一章 緒論 1
一、 腸病毒的分型 1
二、 腸病毒71型的構造與複製 3
三、 腸病毒71型的臨床病徵與流行病學 8
四、 腸病毒71型引起之免疫反應 13
五、 腸病毒71型的毒性決定基因研究 14
六、 腸病毒71型的生活史 17
七、 腸病毒71型之2C蛋白 20
八、 腸病毒71型之3C蛋白 22
九、 病毒之感染性克隆(infectious clone)系統 24
十、 研究動機及目標 25
第二章 材料與方法 28
一、 細胞與病毒 28
二、 感染性克隆(infectious clone)之建構 34
三、 定點突變(site direct mutagenesis) 43
四、 一氧化氮對病毒之影響 46
五、 重組分析(recombination analysis) 49
六、 親緣演化分析 (phylogenetic analysis) 50
七、 蛋白質結構預測軟體SWISS-MODEL 50
八、 統計分析 (Statistical analysis) 50
第三章 結果 51
一、 腸病毒71型臨床分離株之全長序列比較 51
1. 2008年與2012年之腸病毒71型病毒株全長序列的分析 52
2. 1998年大流行之腸病毒71型病毒株全長序列的分析 53
3. 1998年(基因型C2)、2008年與2012年(基因型B5)之腸病毒71型病毒株全長序列的比較 54
二、 腸病毒71型突變株之生物特性 55
1. 生長速率分析 56
2. 病毒溶斑型態分析 57
3. 溫度感受性分析 (temperature sensitivity) 57
4. 細胞凋亡分析 (apoptosis) 58
三、 一氧化氮對突變前後之腸病毒71型的抑制效果 59
1. 亞硝基乙醯青黴胺(SNAP)對細胞的毒性測試 59
2. SNAP在細胞內釋放NO的情形 60
3. 不同時間加入 SNAP 對腸病毒71型生長之影響 60
4. SNAP 對腸病毒71型生長曲線的影響 60
5. SNAP 對腸病毒71型突變株複製之影響 61
第四章 討論 62
參考文獻 70

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系統識別號 U0026-2608201115565100
論文名稱(中文) 長期自主性滾輪運動對下視丘調控心血管表現之影響
論文名稱(英文) Effects of chronic wheel running on the hypothalamic control of cardiovascular performances
校院名稱 成功大學
系所名稱(中) 生理學研究所
系所名稱(英) Department of Physiology
學年度 99
學期 2
出版年 100
研究生(中文) 湯宜禎
學號 s36981052
學位類別 碩士
語文別 中文
口試日期 2011-07-24
論文頁數 78頁
口試委員 指導教授-任卓穎
口試委員-莊季瑛
口試委員-黃阿敏
關鍵字(中) 下視丘
室旁核
γ-胺基丁酸
跑步機運動
滾輪運動
束縛型壓力
心血管反應
一氧化氮合成酶
麩胺酸脫羧酵素67
抑制性離子通道受器聚合蛋白
關鍵字(英) hypothalamus
paraventricular nucleus
γ-aminobutyric acid
treadmill
wheel
immobilization stress
cardiovascular performance
nNOS
glutamic acid decarboxylase 67
gephyrin
學科別分類
中文摘要 許多流行病學統計及研究指出,規律性運動可以降低罹患心血管疾病的風險。下視丘前區的室旁核 ( PVN ) 可以透過神經及賀爾蒙路徑來調控心血管反應,其中在神經路徑的調控上包括興奮性的麩氨酸 ( glutamate ) 以及抑制性的γ-胺基丁酸 ( GABA ) 和一氧化氮 ( NO )。已知大鼠經過長期跑步機運動訓練後,會透過增強下視丘GABA活性來降低心跳與血壓。跑步機及滾輪為現今兩種常用的實驗動物運動模式,而滾輪運動對下視丘調控心血管反應的影響卻是未知的。因此,本實驗欲探討八周自主性滾輪運動訓練對大鼠平靜狀態時及面臨束縛型壓力時心血管反應的影響。五周齡的Wistar大白鼠隨機分成控制組及運動組,利用壓力感測器植入大鼠體內取得即時的心血管參數。除此之外,也分離下視丘前區及後區蛋白質來量化GABA相關蛋白質 ( 一氧化氮合成酶:nNOS、麩胺酸脫羧酵素67:GAD67及抑制性離子通道受器聚合蛋白:gephyrin ) 的表現量。結果顯示自運動滿第二周起運動組平均體重顯著低於控制組,經過八周自主性滾動訓練後,並沒有改善運動組在平靜狀態下的心跳、血壓、自律神經活性及感壓接受器反應活性,除此之外,在面臨束縛型壓力所引起的心血管反應也是無差異的。利用相關係數分析運動組平均跑步距離與心血管參數發現,運動距離與心跳血壓呈現負相關,與脈搏壓則呈現正相關。本篇研究證實,長期自主式滾輪運動並沒有增強大鼠下視丘的GABA系統,且對於平靜及面臨壓力時的心血管表現改善也不大。在下視丘調控心血管方面而言,滾輪運動並不像跑步機運動,對於下視丘調控心血管方面的影響是較小的。
英文摘要 Generally speaking, physical exercise is associated with reduced risk of cardiovascular disease. Hypothalamic paraventricular nucleus (PVN) plays an important role in regulating cardiovascular responses via neuronal and hormonal pathways. The synaptic control of the PVN involves excitatory glutamate and inhibitory γ-aminobutyric acid (GABA) and nitric oxide. Recent results from our laboratory indicated that chronic treadmill running in rats induced hypothalamic adaptations to reset the resting HR/BP to lower levels. Since treadmill running and wheel running are the two most common used animal exercise models, whether and how wheel running exerts similar cardiovascular effects are unknown. In this study, we investigated the effects of eight weeks voluntary wheel running on resting HR/BP and on stress-induced cardiovascular responses in conscious rats. A telemetric probe was inserted into the abdominal aorta of certain animals to monitor their cardiovascular performances real time under resting or immobilization-stressed conditions. In addition, hypothalamic tissues were obtained for quantifying the amount of GABA system-related proteins (such as GAD67, nNOS, and gephyrin). The results showed that since exercise for 2 weeks, exercise group had lower body weight gain than sedentary group. Eight weeks wheel running apparently did not change the resting HR, BP, autonomic nerve activity, baroreceptor reflex sensitivity and hypothalamic GABA system-related protein expression, nor did it ameliorate the immobilization stress-evoked cardiovascular responses. Pearson correlation showed that the running distance were negative correlation with resting HR/BP and positive correlation with pulse pressure. These results indicated that wheel running unlike treadmill running, might exert only minimal effects on the hypothalamic control of cardiovascular performances.
論文目次 中文摘要 ( Abstract in Chinese )--------------------------------------------------- 2
英文摘要 ( Abstract in English ) --------------------------------------------------- 4
致謝 ------------------------------------------------------------------------------------ 6
目錄 ------------------------------------------------------------------------------------- 7
圖表目錄 ------------------------------------------------------------------------------ 8
附圖表目錄 --------------------------------------------------------------------------- 9
縮寫表 ( Abbreviation ) ------------------------------------------------------------ 11
第一章 緒論 ( Introduction ) ----------------------------------------------------- 12
第二章 材料與方法 ( Materials and Methods ) ------------------------------- 18
一、實驗動物 …………………………………………………………………… 18
二、運動模式及居住方式 ……………………………………………………… 18
三、藥品配置 …………………………………………………………………… 19
四、遙測系統血壓量測 ………………………………………………………… 23
五、急性壓力模式 ……………………………………………………………… 26
六、西方點墨法 ………………………………………………………………… 27
七、雙重免疫螢光染色法………………………………………………………… 31
八、統計分析 …………………………………………………………………… 34
第三章 結果 ( Results ) --------------------------------------------- ------------- 35
第四章 討論 ( Discussion ) ------------------------------------------------------- 39
第五章 參考文獻 ( References ) ------------------------------------------------ 47
第六章 表與圖 ( Tables and Figures ) ------------------------------------------ 52
第七章 附錄 ( Appendix ) -------------------------------------------------------- 63
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------------------------------------------------------------------------ 第 19 筆 ---------------------------------------------------------------------
系統識別號 U0026-2707201115491400
論文名稱(中文) 鑑定癌症發展過程中胺基酸代謝的改變
論文名稱(英文) Identification of Altered Amino Acid Metabolisms during Cancer Progression
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) of Biochemistry and Molecular Biology
學年度 99
學期 2
出版年 100
研究生(中文) 喬仲傑
學號 s16981113
學位類別 碩士
語文別 中文
口試日期 2011-07-13
論文頁數 139頁
口試委員 指導教授-賴明德
口試委員-張文粲
口試委員-陳昌熙
關鍵字(中) 癌症
胺基酸
代謝
內質網壓力
雌性激素受體
關鍵字(英) cancer
amino acid
metabolism
ER stress
estrogen receptor
學科別分類
中文摘要 在癌症發展的過程中,癌細胞必須要改變或持續激活代謝路徑上的調控,才能達到足夠的營養供應,滿足癌細胞快速分裂的特性時對於能量上的龐大需求,約略在1950年代,癌症科學家就已經確立麩醯胺酸對於提供癌細胞作為其生長所必須能量的重要性-麩醯胺酸可以作為癌細胞快速分裂時所需之脫氧核糖核酸合成的原料 ; 麩醯胺酸也可促進NADPH的合成,NADPH是脂質的合成中,不可或缺的電子提供者 ; 麩醯胺酸也可以經由電子傳遞鏈產生癌細胞所需ATP及其他非必須胺基酸的合成,幫助構築癌細胞發展所需的大分子產物的合成。此外,也在1950年,科學家們在具有淋巴瘤的老鼠身上,釐清了天門冬氨酸的分解酵素-天門冬氨酸酶,對於有抑制淋巴瘤的效果,並將此發現用於臨床病人身上,在比較有施予天門冬氨酸酶以及沒有施予的急性淋巴白血病的病人之中,結果指出有施予的病人具有較高比例的治癒並回復急性淋巴白血病的病情。除了這兩個胺基酸之外,其他人體中的胺基酸代謝,並沒有在癌症發展的過程中被明確的定位,因此,我們選用了兩個在肝癌以及乳癌的發生過程中扮演重要角色的調節因素-內質網壓力以及雌性激素受體活化,來做為觀察胺基酸代謝在癌症發展過程中的改變,我們選定了胺基酸代謝上的速率決定步驟酵素,或是參與著重要生理意義的合成以及分解的酵素,在我們初步的結果中,我們發現當處理內質網壓力誘導物tunicamycin時,肝癌細胞株:Huh-7中的半胱氨酸及絲氨酸的生合成酵素的mRNA表現量會顯著的上升,同時,精氨酸、組氨酸、脯氨酸、苯丙氨酸及支鏈氨基酸的分解酵素的mRNA,也會在內質網壓力存在下顯著的上升 ; 另一方面,當我們處理雌性激素至乳癌細胞株:MCF-7,活化其雌性激素受體時,我們也可以發現絲氨酸及甲硫氨酸合成酵素有mRNA有顯著上升的情形,同時也伴隨著精氨酸、苯丙氨酸和酪氨酸的分解酵素mRNA上升。除了細胞的實驗外,我們同時也在帶有雌性激素受體病人的乳癌檢體之中驗證了這樣的現象,這樣的結果顯示了當癌細胞處於不同的促進癌細胞生長的環境下,會呈現不同的對於胺基酸代謝及合成的調控,幫助其生長發育 ; 同時,而我們也可以針對這些發生抗的胺基酸代謝做進一步的釐清,找出在不同情形所誘導的癌症發展的過程中有可能作為治療標的胺基酸代謝路徑,有朝一日,能作為日後治療癌症的新策略。
英文摘要 During tumor progression, metabolic switch is required for constitutively activating the uptake and metabolism of nutrients that facilitate cancer cell survival and fuel them growth. Since 1950s cancer biologists have recognized the significance of glutamine (Q) as a tumor nutrients, including nucleotide biosynthesis, which is needed for replicating its genome, NADPH production, which is an indispensable electron donor for lipid synthesis, ATP generation and other nonessential amino acids biosynthesis. Meanwhile, the tumor-inhibitory properties of asparaginase (ASNase) were observed in lymphoma-bearing mice in 1950. Clinical studies have demonstrated that patient with acute lymphoblastic leukemia (ALL) treated with ASNase have higher recovery efficiency than ALL patients did not treat with this drug. Except for the two amino acids, the roles of the other amino acids are not fully elucidated in cancer progression. For that reason, we want to clarify whether the expression of amino acid metabolic genes is altered in different carcinogenic progress such as endoplasmic reticulum (ER) stress, hormone receptor activation, overexpression/activation of Her2/Neu and further to define the relationship between them. According to the bioinformatics wedsites survey, We choose genes that participate in rate-limiting steps and/or play biological significances of amino acid metabolism (ana- or/and cata-bolic). We found that the genes of cysteine biosynthesis were predominantly elevated, and catabolic genes of arginine, histidine, proline, phenylalanine and branched-chain amino acids (BCAAs) were upregulated in ER stress-treated Huh-7 hepatocellular carcinoma cells.On the other hand, expression of anabolic genes of serine and methionine were upregulated and catabolic genes of arginine, phenylalanine and tyrosine metabolism were significantly increased when estrogen receptor-positive MCF-7 breast cancer cells were treated with estrogen. Consistent with our in vitro studies, estrogen receptor-positive clinical breast cancer patient specimens show the same expression pattern of anabolic genes of serine and methionine and catabolic genes of arginine, phenylalanine and tyrosine metabolism. These results suggested that amino acid metabolisms are reprogrammed in the different context of cancer, and we will focus on the influence of this altered amino acid metabolisms during cancer progression in detail and try to find potential candidates for cancer therapy in the future.
論文目次 中文摘要..............................................Ⅰ
英文摘要..........................................Ⅲ
誌謝...............................................................................................................................................................Ⅴ
目錄...............................................................................................................................................................Ⅶ圖目錄..........................................................................................................................................................Ⅹ
附圖目錄.................................................................................................................................................ⅩⅢ
附表目錄.................................................................................................................................................ⅩⅥ

緒論......................................................................................................................................1
1.1 癌細胞中能量代謝的異常調控:醣類與脂肪路徑的改變........... 1
1.2 內質網壓力與肝癌的發生............................4
1.3 雌性激素與乳癌發展之的相關性..........7
1.4 胺基酸代謝與癌症發展相關性...........10
1.5 研究目標與策略............................13

材料與方法.....16
2.1 實驗材料與溶液製備................................................................................16
2.2 細胞培養與藥物處理 (Cell culture and treatment)........................18
2.3 質體製備 (Plasmid preparation)............................................................21
2.4 抽取細胞RNA..............................................................................................24
2.5 聚合酶連鎖反應..........................................................................................26
2.6 西方墨點法...................................................................................................30
2.7 繪圖軟體與統計分析................................................................................37

實驗結果.......................................................................................................................38
3.1 內質網壓力會造成胺基酸代謝基因的改變.....................................38
3.2 雌性激素受體活化會造成胺基酸代謝基因的改變......................40
3.3 PSAT1、PSPH、GLDC、ARG2、TH、GLS2、MAT1A2的mRNA

在臨床乳癌組織中有過度表現的情形................................................42

討論....................................................................................................................................43圖..........................................................................................................................................53
參考文獻.......................................................................................................................78

附圖與附表.................................................................................................................96
自述..................................................................................................................................139


圖目錄
Figure 1. 內質網壓力 (ER stress)誘導物Tunicamycin (TM)能成功促進GRP78
mRNA增加................................................................................................................................................53
Figure 2. 在內質網壓力(ER stress)下會促進cysteine的合成酵素CBS和CTH的mRNA表現量增加................................................................................................................................54
Figure 3. 在內質網壓力(ER stress)下會促進serine的合成酵素PHGDH及、PSAT1及PSPH的mRNA表現量增加...........................................................................................55
Figure 4. 在內質網壓力(ER stress)下會促進glycine的合成酵素SHMT2的mRNA表現量增加...............................................................................................................................................56
Figure 5. 在內質網壓力(ER stress)下會促進proline的合成酵素PYCR2和分解酵素PRODH的mRNA表現量增加.....................................................................................................57
Figure 6. 在內質網壓力(ER stress)下會促進histidine分解的酵素HAL的mRNA表現量增加...............................................................................................................................................58
Figure 7. 在內質網壓力(ER stress)下會促進phenylalanine的分解酵素TAT的mRNA表現量增加.................................................................................................................................59
Figure 8. 在內質網壓力 (ER stress)下會促進glutamine的分解酵素GLS2及glutamate的分解酵素GLUD1的mRNA表現量增加.............................................................60
Figure 9. 在內質網壓力(ER stress)下會促進arginine合成的酵素ASL及分解的酵素ARG2的mRNA表現量增加....................................................................................................61
Figure 10. 在內質網壓力(ER stress)下會促進BCAA的分解酵素BCAT1的mRNA表現量增加...............................................................................................................................................62
Figure 11. 在內質網壓力 (ER stress)下不會改變methionine合成、分解及其他相關的代謝酵素的mRNA表現量.......................................................................................................63
Figure 12. 在內質網壓力(ER stress)下不會改變tryptophan的分解酵素TDO2及TPH2的mRNA表現量..........................................................................................................................64
Figure 13. 雌性激素 (Estradiol)會促進progesterone receptor (PGR)的mRNA表現量增加及下游基因的protein表現量增加............................................................................65
Figure 14. 雌性激素 (Estradiol)會促進tyrosine的分解酵素TH的mRNA表現量增加...............................................................................................................................................................66
Figure 15. 雌性激素 (Estradiol)會促進serine的合成酵素PSAT1、PSPH的mRNA表現量增加...............................................................................................................................................67
Figure 16. 雌性激素 (Estradiol)會促進glycine的分解酵素GLDC的mRNA表現量增加.........................................................................................................................................................68
Figure 17. 雌性激素 (Estradiol)會促進arginine的分解酵素ARG2的mRNA表現量增加.........................................................................................................................................................69
Figure 18. 雌性激素 (Estradiol)會促進methionine的分解酵素MAT1A的mRNA表現量增加..............................................................................................................................................70
Figure 19. 雌性激素 (Estradiol)會促進glutamine的分解酵素GLS2的mRNA表現量增加.........................................................................................................................................................71
Figure 20. 雌性激素 (Estradiol)會降低tryptophan的分解酵素TPH2的mRNA表現量...............................................................................................................................................................72
Figure 21. 雌性激素 (Estradiol)不會改變cysteine的合成酵素CBS和CTH的mRNA表現量...........................................................................................................................................73
Figure 22. 雌性激素 (Estradiol)不會改變BCAA的分解酵素的mRNA表現量....74
Figure 23. 雌性激素 (Estradiol)不會改變proline的合成酵素PYCR2及分解酵素PRODH的mRNA表現量....................................................................................................................75
Figure 24. PSAT1、PSPH、GLDC、MAT1A、GLS2及ARG2基因表現量在具有雌性激素受體的臨床乳癌組織中皆有過度表現的情形.................................................76

附圖目錄
附圖1. Cysteine代謝路徑..................................................................................................................96
附圖2. Serine-Glycine代謝路徑.....................................................................................................97
附圖3. ProlineArginine代謝路徑...................................................................................................98
附圖4. Histidine代謝路徑.................................................................................................................99
附圖5. Phenylalanine/Tyrosine代謝路徑.................................................................................100
附圖6. Glutamine/Glutamate代謝路徑....................................................................................101
附圖7. Arginine代謝路徑...............................................................................................................102
附圖8. BCAA代謝路徑....................................................................................................................103
附圖9. Methionine代謝路徑..........................................................................................................104
附圖10. Tryptophan代謝路徑......................................................................................................105
附圖11. ER stress的活化可能會促進細胞內glutathionine (GSH)含量上升..........106

附圖12. ER stress的活化可能會促進細胞內ATP生成、GSH的含量增加及造成細胞生長和細胞凋亡.............................................................................................................................107

附圖13. ER stress的活化可能會促進細胞內2-氧化戊二酸 (2-oxoglutarate)及glutamate的含量增加........................................................................................................................108

附圖14. ER stress的活化可能會促進細胞內苯丙酮酸 (phenylpyruvate)的含量增加............................................................................................................................................................109

附圖15. ER stress的活化可能會促進細胞內脂質合成增加.......................................110

附圖16. 內質網壓力和胺基酸飢餓能夠針對相同的下游進行轉錄活化............111

附圖17. Estrogen receptor的活化可能會促進細胞內DNA的合成及增加DNA甲基化.......................................................................................................................................................112

附圖18. Serine-glycine代謝路徑能促進purines及pyrimidines的合成................113

附圖19. Estrogen receptor的活化可能會促進細胞內的尿素循環 (urea cycle) ..........................................................................................................................................................114


附圖20. Estrogen receptor的活化可能會促進細胞內黑色素 (melanin)的含量..................................................................................................................................................................115


附圖21. 比對核酸微陣列的數據,GRP78的表現量在具有內質網壓力的情形下有顯著增加的現象........................................................................................................................116


附圖22. 比對核酸微陣列的數據,CTH及CBS的表現量在具有內質網壓力的情形下有顯著增加的現象..............................................................................................................117


附圖23. 比對核酸微陣列的數據,PHGHD、PSAT1及PSPH的表現量在具有內質網壓力的情形下有顯著增加的現象..............................................................................118

附圖24. 比對核酸微陣列的數據,SHMT2的表現量在具有內質網壓力的情形下有顯著增加的現象........................................................................................................................120

附圖25. 比對核酸微陣列的數據,BCAT1的表現量在具有內質網壓力的情形下有顯著增加的現象........................................................................................................................121

附圖26. 比對核酸微陣列的數據,TAT的表現量在具有內質網壓力的情形下有顯著增加的現象.............................................................................................................................122

附圖27. 比對核酸微陣列的數據,PYCR2的表現量在具有內質網壓力的情形下有顯著增加的現象,而PRODH則沒有..............................................................................123

附圖28. 比對核酸微陣列的數據,HAL的表現量在具有內質網壓力的情形下沒有顯著增加的現象........................................................................................................................124

附圖29. 比對核酸微陣列的數據,GLUL、GLUD1及GLS2的表現量在具有內質網壓力的情形下有顯著增加的現象....................................................................................125

附圖30. 比對核酸微陣列的數據,ASL及ARG2的表現量在具有內質網壓力的情形下有顯著增加的現象........................................................................................................127

附圖31. 比對核酸微陣列的數據,PGR的表現量在雌性激素受體活化的情形下有顯著增加的現象........................................................................................................................128

附圖32. 比對核酸微陣列的數據,PSAT1及PSPH的表現量在雌性激素受體活化的情形下有顯著增加的現象...................................................................................................129

附圖33. 比對核酸微陣列的數據,GLS2表現量在雌性激素受體活化的情形下有顯著增加的現象.............................................................................................................................130

附圖34. 比對核酸微陣列的數據,ARG2表現量在雌性激素受體活化的情形下有顯著增加的現象.............................................................................................................................131

附圖35. 比對核酸微陣列的數據,TH表現量在雌性激素受體活化的情形下有顯著增加的現象..................................................................................................................................132

附圖36. 比對核酸微陣列的數據,GLDC表現量在雌性激素受體活化的情形下有顯著增加的現象.............................................................................................................................133

附圖37. 比對核酸微陣列的數據,MAT1A表現量在雌性激素受體活化的情形下沒有顯著增加的現象...................................................................................................................134


附表目錄

附表1. 選定之胺基酸代謝基因及相對應之酵素縮寫簡表.........................................135
附表2. 選定之胺基酸代謝之分解及合成基因全名與縮寫對應..............................136
附表3. 在內質網壓力及雌性激素受體活化情形下表現量會有所改變的胺基酸代謝酵素縮寫表..................................................................................................................................138
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系統識別號 U0026-3107201221064400
論文名稱(中文) 製備嵌段共聚胺酸與雙醣修飾聚胺酸及礦化之自組裝高分子載體應用於藥物傳輸
論文名稱(英文) Preparation of block copolypeptides and glycopeptides and mineralization of self-assembled glycopeptides carriers for drug delivery
校院名稱 成功大學
系所名稱(中) 化學工程學系碩博士班
系所名稱(英) Department of Chemical Engineering
學年度 100
學期 2
出版年 101
研究生(中文) 陳雲耑
學號 N36994467
學位類別 碩士
語文別 中文
口試日期 2012-07-06
論文頁數 105頁
口試委員 指導教授-詹正雄
口試委員-張鑑祥
口試委員-侯聖澍
口試委員-林宏殷
關鍵字(中) 聚胺基酸
分子自組裝
醣類
藥物傳輸
點擊化學
關鍵字(英) Polypeptides
self-assembly
saccharides
drug delivery
click chemistry
學科別分類
中文摘要 於本研究中,經由胺基酸N-carboxyanhydrides (NCAs)的開環聚合反應(Ring-opening polymerization)我們製備得到嵌段共聚胺基酸, poly(γ-benzyl-L -glutamate)-block-poly(L-phenylalanine) (PBLG-b-PPhe)與poly(γ-benzyl-L -glutamate)-block-polyglycine (PBLG-b-PGly)。在移除PBLG嵌段上的benzyl保護基後,我們得到水溶性的雙親性嵌段共聚胺基酸, poly(L-glutamic acid)-block-poly(L-phenylalanine) (PLGA-b-PPhe)與poly(L-glutamic acid)-block -polyglycine (PLGA-b-PGly),接著經由點擊化學的銅催化炔-疊氮環化加成反應(Click chemistry:Copper-catalyzed Azide-Alkyne Cycloaddition),我們成功地製備得到具有雙醣修飾的雙親性胺基酸嵌段共聚物自組裝結構,Lactobionic acid-block-poly(L-glutamic acid)-block-poly(L-phenylalanine) (Lac-b-PLGA-b-PPhe)與Lactobionic acid-block-poly(L-glutamicacid)-block -polyglycine (Lac-b-PLGA-b-PGly)。本雙親性胺基酸嵌段共聚物於水溶液中會自組裝形成微胞(Micelles)結構,在經由乳醣酸的修飾並且經由Calcium Phosphate (CaP)的礦化沉積後,此具有半乳醣基之雙親性胺基酸嵌段共聚物形成的微胞可以成為具有標定肝癌細胞功能的藥物載體。
在嵌段共聚胺基酸,PBLG-b-PPhe與PBLG-PGly,的組成以及結構性質分析上,我們使用了凝膠滲透層析儀(GPC)、紅外線光譜儀(FT-IR)、熱分析儀(DSC)、廣角度X光散射(WAXS)以及核磁共振光譜儀(13C NMR 與1H NMR)等儀器鑑定。接下來,藉由動態光散射粒徑分析儀(DLS)、穿透式電子顯微鏡(TEM)、螢光光譜儀(PL)以及原二色光譜儀(CD)的測定,分析探討本雙親性胺基酸嵌段共聚物在水溶液中自組裝的結構及性質。隨後我們利用雙醣修飾的雙親性胺基酸嵌段共聚物微胞包覆癌症藥物Doxorubicin (DOX)以及進行表面改質礦化,使成為良好的標靶藥物載體,並且透過紫外光-可見光光譜儀(UV)偵測載體內藥物的釋放速率。在未來,我們會進一步研究本實驗製備得到的具有半乳醣基之雙親性胺基酸嵌段共聚物的細胞毒性及細胞吞噬機制。
英文摘要 In the present study, poly(γ-benzyl-L-glutamate)-block-poly(L-phenylalanine) (PBLG-b-PPhe) and poly(γ-benzyl-L-glutamate)-block-polyglycine (PBLG-b -PGly) were synthesized through the ring-opening polymerization (ROP) of N-carboxyanhydrides (NCAs) of the corresponding α-amino acids. The protecting γ-benzyl groups on PBLG block were then removed to give poly(L-glutamic acid) -block-poly(L-phenylalanine) (PLGA-b-PPhe) and poly(L-glutamic acid)-block -polyglycine (PLGA-b-PGly) amphiphilic block copolypeptides. Afterwards, the sugar-containing amphiphilic block copolypeptides self-assembled structures, Lactobionic acid-block-poly(L-glutamic acid)-block-poly(L-phenylalanine) (Lac -b-PLGA-b-PPhe) and Lactobionic acid-block-poly(L-glutamic acid)-block -polyglycine (Lac-b-PLGA-b-PGly), were obtained via Click chemistry:Copper -catalyzed Azide-Alkyne Cycloaddition (CuAAC). The as-prepared amphiphilic block copolypeptides, PLGA-b-PPhe and PLGA-b-PGly can self-assemble to form micelles. With the incorporation of sugar moiety, the micelles can be the targeted drug carriers toward liver cancer cells.
The conformation and structure of the obtained block copolypeptides were characterized by gel permeation chromatography (GPC), Fourier transform -infrared spectroscopy (FT-IR), Differential Scanning Calorimetry (DSC), wide-angle X-ray scattering (WAXS), carbon nuclear magnetic resonance (13C NMR) spectroscopy and proton nuclear magnetic resonance (1H NMR) spectroscopy. The particle size, morphology, and chain conformation of the aggregates in aqueous solutions were determined by transmission electron microscopy (TEM), dynamic light scattering (DLS), and circular dichroism (CD), respectively. Then, we encapsulated doxorubicin (DOX), the drug used in cancer therapy, and mineralized with calcium phosphate to improve the efficacy of the carriers. The drug release from the carriers was evaluated by UV-vis spectroscopy. In the future, the cell cytotoxicity and cellular uptake of the assembled structures used as tumor-targeted carriers for drug delivery will be investigated.
論文目次 摘要 I
Abstract III
誌謝 V
第一章 緒論 1
第二章 文獻回顧 4
2.1 開環聚合嵌段共聚胺酸的研究進展 4
2.1.1 嵌段共聚胺酸膠體 4
2.1.2 嵌段共聚胺酸微胞及囊胞 6
2.1.3 共聚胺酸對無機材料的表面改質、結構形成與生長 8
2.1.4 功能化嵌段共聚胺基酸 10
2.2 胺基酸的聚合 11
2.2.1 NCAs的合成 12
2.2.2 胺基酸NCAs開環聚合 13
2.3 點擊化學(Click chemistry) 16
2.3.1 銅催化炔-疊氮環化加成反應(Copper-catalyzed azide - alkyne cycloaddition) (CuAAC) 18
2.4 雙親性嵌段共聚胺酸的自組裝及製備 20
2.5 聚胺基酸作為藥物載體的腫瘤標靶治療 23
2.5.1 醫療用奈米微粒簡介 24
2.5.2 克服奈米微粒在藥物傳輸上的阻礙的策略 25
2.5.3 細胞標的藥物運送 27
2.5.4 藥物的釋放 28
2.5.5 奈米微粒的設計 28
第三章 實驗方法與步驟 32
3.1 實驗藥品 32
3.2 實驗儀器 33
3.3 乳醣酸修飾之雙親性胺基酸嵌段共聚物的合成製備 33
3.3.1 Azide-Lac的合成 35
3.3.2 嵌段共聚胺基酸的合成(Scheme 3.1 D) 36
3.3.3 雙親性胺基酸嵌段共聚物自組裝結構的雙醣修飾 38
3.4 奈米粒子的製備 38
3.4.1 PLGA-b-PPhe系列 及PLGA-b-PGly系列奈米粒子的配製 38
3.4.2 奈米粒子的礦化 39
3.4.3 奈米粒子藥物的包覆及其礦化 39
3.5 藥物釋放 40
3.6 檢測儀器 40
3.6.1 超導核磁共振光譜儀 40
3.6.2 凝膠滲透層析儀 41
3.6.3 固態核磁共振光譜儀 43
3.6.4 廣角X光繞射儀 45
3.6.5 穿透式電子顯微鏡 45
3.6.6 反射式紅外線光譜儀 46
3.6.7 紅外線光譜儀 47
3.6.8 圓二色光譜儀 47
3.6.9 熱重分析儀 49
3.6.10 差示掃描量熱儀 50
3.6.11 動態光散射偵測儀 53
第四章 結果與討論 57
4.1 乳糖酸修飾之雙親性胺基酸嵌段共聚物的合成及結構鑑定 57
4.1.1 乳醣酸羧酸基的官能化 57
4.1.2 雙親性胺基酸嵌段共聚物的合成 60
4.1.3 點擊化學: 銅催化炔-疊氮環化加成反應(CuAAC) 64
4.2 PBLG-b-PPhe嵌段共聚物結構的分析與鑑定 68
4.2.1 13C NMR分析 68
4.2.2 FT-IR分析 73
4.2.3 WAXS分析 73
4.2.4 DSC分析 75
4.3 雙親性嵌段共聚物自組裝結構的分析鑑定 77
4.3.1 CD分析 77
4.3.2 DLS以及TEM分析 81
4.3.3 TGA分析 92
4.4 Doxorubicin的包覆 93
第五章 結論 95
第六章 參考文獻 97

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