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系統識別號 U0026-0605201113473500
論文名稱(中文) 經磺酸化/磷酸化改質之幾丁聚醣薄膜其及表面特性、血液相容性與細胞相容性之研究
論文名稱(英文) Surface analysis, blood compatibility and cell proliferation evaluations of sulfonated/ phosphorylated chitosan membrane
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) 葉錫誼
研究生(英文) Hsi-Yi Yeh
學號 n3891109
學位類別 博士
語文別 中文
論文頁數 113頁
口試委員 指導教授-林睿哲
口試委員-陳志勇
口試委員-許梅娟
口試委員-李澤民
口試委員-徐善慧
口試委員-蔡偉博
中文關鍵字 幾丁聚醣  磺酸化  保護基  磷酸化  表面電性  聚電解質複合膜  血液相容性  脂肪幹細胞 
英文關鍵字 Sulfonation  chitosan  amino group protection  zeta potential  streaming potential  phosphorylation  polyelectrolyte  blood compatibility  adipose-derived stem cell 
學科別分類
中文摘要 幾丁聚醣(Chitosan)具有良好的生物相容性和生物降解性,在自然界中的含量僅次於纖維素,結構上近似於胺基葡聚醣(glycoaminoglycan,GAGs),胺基葡聚醣是生物體中細胞外基質的主要成分之一。與胺基葡聚醣相較,幾丁聚醣成本較低廉,因此逐漸被應用在組織工程的領域中。由於幾丁聚醣分子上帶正電的胺基(amino group)被認為是幾丁聚醣在細胞培養上獨特生化特性的主要因素,同時研究指出若是能導入磺酸根官能基或是磷酸根官能基將有助於特定細胞的生長。
因此本研究嘗試在幾丁聚醣第六個碳的-OH上導入磺酸根官能基,來模仿細胞外基質中低分子量胺基葡聚醣的結構。同時我們建立起幾丁聚醣第二個碳上一級胺基的保護與去保護策略,希望改質後的幾丁聚醣試片也能保有一些原有的生化特性。
表面分析和血小板吸附測試的結果,證明本研究已經得到具有胺基保護策略的磺酸化幾丁聚醣薄膜,並具有促進血小板吸附,但不會引起凝血活化反應的特性,可以提供後續研究進行進一步的組織工程應用探討。
目前很少人將磺酸根與磷酸根同時導入幾丁聚醣表面,並探討其生化特性。因此本研究進一步製備水溶性的磺酸化幾丁聚醣,並與未改質幾丁聚醣形成聚電解質複合膜(polyelectrolyte complex),再利用表面接枝技術導入磷酸根官能基,使改質後的聚電解質薄膜表面同時含有磺酸根官能基與磷酸根官能基。
由表面分析儀器ESCA以及ATR-FTIR的結果,顯示磺酸根及磷酸根官能基已經成功接枝於幾丁聚醣上。而在血液相容性方面,磷酸化後的幾丁聚醣聚電解質薄膜相較於僅含磺酸根的聚電解質薄膜有縮短血漿再鈣化時間的特性。血小板吸附實驗顯示,單純磷酸化的幾丁聚醣表面具有最高的血小板凝血活化反應,但引進磺酸根官能基後可以增加血小板的相容性。脂肪幹細胞生長實驗則顯示含有磷酸根的表面有促進細胞生長的效果。若試片同時含有磺酸根與磷酸根在一定的比例下,可使試片同時具有增進細胞生長和抑制凝血的特性,對生醫材料上的應用有很大的潛力。
英文摘要 Many researches tried to look for the application of chitosan in tissue engineering since its structure is similar to glycoaminoglycans, the main components of extracellular matrix. Previous studies had indicated that the incorporation of sulfonic or phosphonic functionalities onto chitosan structure would be beneficial to the growth of certain cells. However these modifications almost carried out on the reactive amino groups that were thought as the major character resulting in special biological properties associated with chitosan.
In the first part of this study, an amino group protection- deprotection strategy was explored for surface sulfonation of chitosan membrane with an aim to imitate GAGs’ structures. Various surface chemical characterization results as well as surface zeta potential measurement have indicated that both sulfonate/sulfonic and amino functionalities were coexistent on the deprotected sulfonated chitosan specimen. In vitro platelet adhesion testing has shown that such a deprotected sulfonated chitosan membrane can increase the amount of platelet adhesion while keep those adhered remained unactivated.
Furthermore, in the second part of this study, we phosphorylated the surface of polyelectrolyte film which is composed of chitosan and water soluble sulfonated chitosan. We aimed at the incorporation of phosphonic and sulfonic functionalities onto the film surface. Until now, there is no study even explored the effect of incorporation of both abovementioned anionic functionalities onto the chitosan structure.
Surface analyses by ESCA and ATR-FTIR have shown that these two functional groups have been successfully grafted onto the surface. And the ratio of P/S was dependent upon the weight ratio of phosphorylation agents added.
Blood compatibility evaluation indicated that phosphorylated polyelectrolyte complexes extended the plasma recalcification time as compared to the non-treated chitosan and direct-phosphorylated chitosan film. In addition, these phosphorylated polyelectrolyte complexes showed similar or slightly less platelet-reactivity than the non-phosphorylated counterpart. In contrast, significant platelet activation and adhesion were observed on the direct-phosphorylated chitosan. This implicated that the incorporation of sulfonic acid onto the phosphorylated surface could increase the platelet compatibility. Adipose-derived stem cell incubation study has demonstrated that the incorporation of both phosphonic and sulfonic acid functionalities onto the chitosan surface can enhance the growth of stem cell. Therefore, the phosphorylated polyelectrolyte complexes were not only blood compatible but also stem cell compatible. Consequently, it could be a novel biomaterial in tissue engineering application.
論文目次 目錄

中文摘要……………………………………………………………………I
英文摘要…………………………………………………………………III
誌謝…………………………………………………………………………V
目錄………………………………………………………………………VI
表目錄 …………………………………………………………………XII
圖目錄…………………………………………………………………XIII

第一章 緒論 ………………………………………………………………1

第二章 文獻回顧…………………………………………………4
2.1 組織工程簡介………………………………………………………4
2.1.1細胞的來源……………………………………………………5
2.1.2.細胞支架 ……………………………………………………8
2.1.3培養環境 ……………………………………………………10
2.2 幹細胞介紹 ………………………………………………………12
2.2.1 胚胎幹細胞…………………………………………………12
2.2.2 成體幹細胞…………………………………………………13
2.3 細胞支架的材料 …………………………………………………16
2.3.1 人造合成高分子……………………………………………16
2.3.1.1聚乙二醇酸/聚乳酸 …………………………………17
2.3.1.2聚己內酯多元醇………………………………………19
2.3.2 天然高分子…………………………………………………20
2.3.2.1 膠原蛋白………………………………………………21
2.3.2.2 胺基葡聚糖……………………………………………23
2.3.2.3 玻尿酸…………………………………………………25
2.4 幾丁聚醣……………………………………………………26
2.4.1 幾丁聚醣的結構……………………………………………26
2.4.2 幾丁聚醣的化學特性………………………………………27
2.4.3 幾丁聚醣的生物降解性和生物相容性……………………28
2.4.4 幾丁聚醣的生化特性………………………………………28
2.4.5 幾丁聚醣在組織工程上的應用……………………………29

第三章 實驗藥品與儀器簡介…………………………………………31
3.1 實驗藥品…………………………………………………………31
3.2 實驗儀器…………………………………………………………37
3.3 儀器原理簡介……………………………………………………39
3.3.1 化學分析電子光譜儀………………………………………39
3.3.2 減弱式全反射傅立葉紅外線光譜儀………………………40
3.3.3 靜態接觸角…………………………………………………41
3.3.4 薄膜界面電位分析儀………………………………………42

第四章 具胺基保護策略之表面磺酸化幾丁聚醣薄膜………43
4.1 研究動機 ……………………………………………… 43
4.2 研究目的………………………………………………44
4.3 實驗方法………………………………………………45
4.3.1 幾丁聚醣薄膜的製備………………………………………45
4.3.2具胺基保護策略的幾丁聚醣表面磺酸化改質……………46
4.3.2.1胺基保護反應…………………………………………46
4.3.2.2表面磺酸化反應………………………………………46
4.3.2.3去保護基反應…………………………………………46
4.3.3表面胺基的定量……………………………………………47
4.3.4表面性質的量測……………………………………………47
4.3.5表面電性量測………………………………………………48
4.3.6 血液相容性測試……………………………………………48
4.3.6.1血漿再鈣化凝固時間…………………………………48
4.3.6.2 血小板吸附實驗………………………………………49
4.4 實驗結果………………………………………………50
4.4.1化學分析電子光譜儀………………………………………50
4.4.2 減弱式全反射傅立葉紅外光譜儀…………………………53
4.4.3 表面胺基定量………………………………………………53
4.4.4 靜態接觸角量測……………………………………………54
4.4.5 表面電性量測………………………………………………55
4.4.6 血漿再鈣化時間……………………………………………57
4.4.7 血小板吸附實驗……………………………………………58
4.5 結果討論…………………………………………………59
4.5.1 幾丁聚醣薄膜表面胺基的保護和去保護策略……………59
4.5.2 幾丁聚醣薄膜表面磺酸化…………………………………60
4.5.3 改質後幾丁聚醣試片的血液相容性質……………………61

第五章 幾丁聚醣及其磺酸化衍生物之聚電解質薄膜表面磷酸化之探討………………………………………………75
5.1 研究動機…………………………………………………………75
5.2 研究目的…………………………………………………………76
5.3 實驗方法…………………………………………………………77
5.3.1 水溶性磺酸化幾丁聚醣之製備……………………………77
5.3.2試片成膜……………………………………………………78
5.3.2.1 幾丁聚醣薄膜…………………………………………78
5.3.2.2 聚電解質薄膜…………………………………………78
5.3.3薄膜之表面磷酸化…………………………………………79
5.3.4 物性與化性分析……………………………………………79
5.3.4.1 水溶性磺酸化幾丁聚醣之性質分析…………………79
5.3.4.2 薄膜之表面分析………………………………………79
5.3.5血液相容性測試……………………………………………80
5.3.5.1血小板吸附實驗………………………………………80
5.3.5.2血漿再鈣化時間………………………………………81
5.3.6脂肪幹細胞生長測試………………………………………81
5.3.6.1 細胞型態觀察…………………………………………81
5.3.6.2 細胞生長評估…………………………………………82
5.4 實驗結果…………………………………………………………82
5.4.1 水溶性磺酸化幾丁聚醣……………………………………82
5.4.2化學分析電子儀…………………………………………83
5.4.3 減弱式全反射傅立葉紅外光譜儀…………………………84
5.4.4 靜態接觸角量測……………………………………………85
5.4.5 血漿再鈣化時間……………………………………………85
5.4.6血小板吸附實驗……………………………………………86
5.4.7脂肪幹細胞生長測試………………………………………87
5.5 結果討論…………………………………………………………87
5.5.1表面性質分析………………………………………………87
5.5.2 血液相容性…………………………………………………88
5.5.3 細胞生長測試………………………………………………89

第六章 結論與未來展望………………………………………………100

參考文獻…………………………………………………………………104
著作目錄…………………………………………………………………113
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