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系統識別號 U0026-0508201510562800
論文名稱(中文) 台灣鯛魚鱗膠原蛋白-明膠-幾丁聚醣交聯創傷敷料之製備與特性研究
論文名稱(英文) Preparation and characterization of cross-linked Taiwan Tilapia scale collagen–gelatin–chitosan film used in wound treatment
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 103
學期 2
出版年 104
研究生(中文) 劉媛婷
研究生(英文) Yuan-Ting Liu
學號 L36021327
學位類別 碩士
語文別 中文
論文頁數 59頁
口試委員 指導教授-黃福永
口試委員-蕭世裕
口試委員-王小萍
中文關鍵字 膠原蛋白  明膠  幾丁聚醣  戊二醛  交聯 
英文關鍵字 collagen  gelatin  chitosan  glutaraldehyde  cross-linking 
學科別分類
中文摘要 台灣鯛(Taiwan Tilapia)為台灣主要養殖魚類之一,同時也製造了大量的水產廢棄物,而其魚鱗含有豐富的膠原蛋白纖維,因此本研究探討自魚鱗萃取膠原蛋白(collagen)與明膠(gelatin)的方式,並與幾丁聚醣(chitosan)混合,以戊二醛(glutaraldehyde)進行交聯作用,得到適合做為傷口敷料的薄膜。
以胃蛋白酶(pepsin)對魚鱗進行膠原蛋白萃取,測量各種條件所得到的蛋白溶液濃度,並以SDS-PAGE觀察蛋白質分子量分布,而最適合的萃取條件為使用1mg/mL之胃蛋白酶 (10g清洗乾淨的乾的魚鱗泡在pH2的酸溶液約200mL中),於30℃環境中的水解魚鱗24小時得產率為0.86%的膠原蛋白。將10g清洗乾淨的乾的魚鱗泡在200mL的二次水中煮約2小時得到45.7%的明膠。將兩者以重量比1:10配置溶液,而蛋白溶液再以體積比2:1與2%幾丁聚醣混合,得到質地光滑而具有韌性與彈性之薄膜。為了增加薄膜的抗水性與機械強度,則加入了戊二醛作為交聯劑,並以NHN assay檢測不同條件所製成的薄膜交聯程度,以濃度1.0%之戊二醛交聯5小時,可得到最好的交聯指數為48.69%,以及膨潤度為237.11%。定性方面,藉由紅外線光譜(IR)可以看出原本膠原蛋白/明膠/幾丁聚醣的一級胺基吸收峰(1558cm-1和3440cm-1)在經過交聯後明顯縮小,證實三者確實經由戊二醛發生交聯反應。另外也使用圓二色光譜(CD)分析膠原蛋白交聯前後的二級結構,發現膠原蛋白與戊二醛交聯後由原本的α-helix構型逐漸轉往β-sheet與β-turn。最後透過掃描式電子顯微鏡(SEM)觀察交聯前後的蛋白薄膜表面,可以看出隨著交聯程度上升,組織表面逐漸形成完整片狀結構,並以蜂巢狀整齊排列。雖然未交聯的膠原蛋白/明膠/幾丁聚醣薄膜對小鼠傷口癒合能力無明顯幫助,但預期交聯後的薄膜構造將有助於細胞的附著與增生,並對細胞培養與小鼠傷口癒合能力有正面的效果。
英文摘要 Collagen and gelatin obtained from Taiwan Tilapia scales were mixed appropriately with chitosan to form a film as wound dressing. The optimal pepsin-extracted collagen was obtained by using pepsin to extract collagen in pH2 condition for least 24hr. gelatin was obtained by directly extracting under boiling condition. Collagen and gelatin were mixed in to a solution with the weight ratio of 1:10, then the solution was mixed with 2% chitosan with volume ratio of 2:1. After incubating in 30℃ for 5hr followed by lyophilization, a disk film was obtained. Another film was produced by adding glutadialdehyde as a cross-linking reagent to the tri-component solution. The cross-linking index was measured by using ninhydrin assay and the anti-hydrolysis ability was assayed by the swelling ratio. The IR spectra showed the intensity of amide I peak decreased, indicating that the cross-linking reaction between in glutadialdehyde and the –NH2 of collagen took place. Far-UV (ultraviolet) CD spectrum showed that the major 2nd order structure component of the collagen/gelatin changed fromα-helix toβ-sheet andβ-turn gradually after cross-linking. The films surface morphology observed by SEM showed that the cross-linking film exist in a honeycomb structure that would provide an appropriate environment for cell growth and may promote the ability of wound healing. Therefore, we expect cell culture and wound treatment tests with cross-linked film will showed better wound healed ability than none cross-linked film.
論文目次 中文摘要 I
英文摘要 II
誌謝 XIII
目錄 XIV
表目錄 XVII
圖目錄 XVIII
第一章 序論 1
1.1、前言 1
1.2、台灣鯛簡介 2
1.2.1、台灣鯛由來 2
1.2.2、台灣產銷概況 3
1.2.3、魚鱗成分與應用 3
1.3、膠原蛋白簡介 4
1.3.1、膠原蛋白來源 4
1.3.2膠原蛋白的結構 4
1.3.3膠原蛋白的型態與分類 6
1.3.4膠原蛋白的特點和生醫上的應用 7
1.3.5、膠原蛋白之酵素水解 8
1.4、明膠簡介 11
1.4.1、明膠的來源 11
1.4.2、明膠的特性與應用 12
1.5、幾丁聚醣 13
1.5.1幾丁聚醣的來源 13
1.5.2幾丁聚醣的結構與物理化學性質 13
1.5.3幾丁聚醣於生醫產業上的應用 15
1.6、交聯原理與目的 17
1.7、醫用敷料材料簡介 18
1.7.1敷料的特性 18
1.7.2敷料的種類 18
第二章 藥品、設備與實驗方法 20
2.1、實驗材料 20
2.2、實驗方法 22
2.2.1、台灣鯛魚鱗膠原蛋白之前處理 22
2.2.2、台灣鯛魚鱗膠原蛋白之萃取 22
2.2.3、台灣鯛魚鱗明膠之萃取 23
2.2.4、膠原蛋白/明膠溶液與幾丁聚醣複合基材製備 23
2.2.5、膠原蛋白/明膠/幾丁聚醣以戊二醛交聯製成薄膜 23
2.3、分析方法 24
2.3.1、蛋白質濃度測試(Braford assay) 24
2.3.2、一維電泳分析 (SDS page) 24
2.3.3、交聯指數評估(Ninhydrin assay自由胺基測量) 26
2.3.4、膨潤度測試(swelling ratio) 27
2.3.5、紅外線光譜分析(IR Analysis) 27
2.3.6、圓二色光譜分析(Circular Dichroism, CD) 28
2.3.7、掃描式電子顯微鏡之表面型態分析(Scanning electron microscope, SEM) 28
2.3.8、膠原蛋白敷料對小白鼠傷口癒合能力之測試 29
第三章 結果與討論 30
3.1、台灣鯛魚鱗膠原蛋白之前處理 30
3.2、魚鱗膠原蛋白酵素萃取 31
3.3、膠原蛋白/明膠溶液與幾丁聚醣複合基材製備 35
3.4、膠原蛋白/明膠/幾丁聚醣以戊二醛交聯製成薄膜 38
3.5、交聯指數評估(Ninhydrin assay自由胺基測量) 41
3.6、膨潤度測試(swelling ratio) 43
3.7、紅外線光譜分析(IR Analysis) 44
3.8、圓二色光譜分析(Circular Dichroism, CD) 47
3.9、掃描式電子顯微鏡之表面型態分析(Scanning electron microscope, SEM) 50
3.10、膠原蛋白敷料對小白鼠傷口癒合能力之測試 52
第四章 結論 55
第五章 參考文獻 57
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