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研究生:鄭雅云
研究生(外文):Ya yun Cheng
論文名稱:含聚麩胺酸/膠原蛋白之新型生物膠的製備與定性
論文名稱(外文):Preparation and characterization of a new type bioglue made from γ-poly-(glutamic acid) and collagen.
指導教授:蔡曉雯蔡曉雯引用關係
指導教授(外文):Shiao-Wen Tsai
學位類別:碩士
校院名稱:長庚大學
系所名稱:生化與生醫工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:57
中文關鍵詞:組織黏著劑聚麩氨酸膠原蛋白
外文關鍵詞:bioadhesivegamma polyglutamic acidcollagen
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本實驗的主要目的為設計開發以膠原蛋白與聚麩胺酸混合溶液之新型組織黏著劑,探討除了增強流體黏著強度外,藉由膠原蛋白於體外形成D-period微纖維結構對細胞生長的重要影響,結合聚麩氨酸具備的抗菌性及保水性同時可作為組織再生模板,以促進組織器官之修復。
以掃描式電子顯微鏡觀察膠原蛋白與聚麩胺酸共同組成之混合溶液,膠原蛋白纖維可形成D-period之週期性結構排列,證明於適當的環境調控下,γ-聚麩胺酸的存在並不影響膠原蛋白纖維的體外重組。藉由實驗設計法歸論出黏著劑最適組成為 膠原蛋白/γ-聚麩胺酸混合溶液,濃度分別為6、15 mg/ml,以300mg/ml之EDC進行化學交聯,兩者體積比為10:1。黏著劑最大黏著強度於交聯後8分鐘內可達437±40gf/cm2,於24小時後可增強約為779±76gf/cm2,相較於市售血纖維蛋白組織膠於使用後8分鐘強度約為314±2 gf/cm2,黏著強度明顯增強。經由EDC交聯γ-聚麩胺酸/膠原蛋白混合溶液製備之組織黏著劑,於凝膠狀態下之吸水膨潤比例約為30%,且其重量並不隨著時間而變化,顯示膠體具有相當良好的穩定性。由體外細胞毒性測試發現,以EDC進行交聯之γ-聚麩胺酸/膠原蛋白組織黏著劑具有輕微細胞毒性,但不至於影響細胞分裂增生。而由動物實驗結果可觀察得知,凝膠態組織黏著劑植入大鼠皮下約4週可分解至小於原始體積50%,並於植入8週後可完全分解。藉由病理組織切片觀察發現,僅於植入第7天有輕微發炎反應發生,顯示黏著劑具良好的生物相容性。
The aim of this study is to develop a novel bioadhesive glue composed of γ-PGA and collagen crosslinked with EDC. Except remain the function of homeostasis, we also investigated the ability of glue in improving adhesive strength and tissue regeneration. The use of collagen is attractive since the natural fibrous network with D-periodic unique structure is known to serve as a scaffold for many type of tissue repair. γ-PGA is biodegradable and highly hydrophilic under neutral environment, it could enhance the viscosity of solution.
Under TEM examined, we had confirmed that the D-period structure of collagen fiber remain in γ-PGA/collagen bioadhesive glue. According to the results of Robust design, we chose 15mg/ml of γ-PGA, 6mg/ml of collagen and 300mg/ml of EDC (the volume ratio of γ-PGA/collagen mixed solution to EDC is 10:1.) as the optimum composition of γ-PGA/collagen bioadhesive glue. The binding strength of γ-PGA/collagen glue was about 437±40gf/cm2 within 8 minutes, and increased to 779±76gf/cm2 after 24 hours, that was much stronger than the fibrin glue. The swelling ratio of γ-PGA/collagen glue is about 30% and has no change for further incubated with PBS buffer solution. Cell cytotoxicity test showed that there was a little relative toxic effect of γ-PGA/collagen glue, but the proliferation of L929 cells remained at normal rate. Animal study demonstrated that γ-PGA/collagen glue has well biocompatibility and biodegradation since the glue was completely degraded after 8 weeks of implantation.
中文摘要 i
Abstract iii

第一章 前言 1
1-1 生物組織黏著劑的簡介 1
1-2 研究動機與目的 5

第二章 實驗材料簡介 6
2-1 γ-聚麩胺酸簡介 6
2-2 膠原蛋白簡介 13

第三章 實驗藥品與儀器 20
3-1 實驗藥品 20
3-2 儀器設備 22

第四章 實驗方法 23
4-1膠原蛋白溶液之製備 23
4-1-1 動物性膠原蛋白的萃取純化 23
4-1-2 動物性膠原蛋白之定性試驗 24
4-1-3 動物性膠原蛋白之定量分析試驗 24

4-2 γ-聚麩胺酸/膠原蛋白生物組織黏著劑之製備 27
4-2-1 以EDC交聯γ-聚麩胺酸/膠原蛋白組織黏著劑之製備 27
4-2-2 γ-聚麩胺酸/膠原蛋白生物組織黏著劑微結構之觀察 29
4-2-3 γ-聚麩胺酸/膠原蛋白生物組織黏著劑之黏度測試 29
4-2-4 γ-聚麩胺酸/膠原蛋白生物組織黏著劑之拉伸強度測試 29

4-3 凝膠態γ-聚麩胺酸/膠原蛋白生物組織黏著劑含水率測試 30
4-4 In vitro細胞毒性測試 31
4-5 In vivo動物實驗測試 32

第五章 結果與討論 33
5-1膠原蛋白溶液之製備 33
5-1-1 膠原蛋白純度之定性試驗 33
5-1-2 膠原蛋白溶液濃度之定量試驗 33

5-2 γ-聚麩胺酸/膠原蛋白生物組織黏著劑之製備 33
5-2-1 穿透式電子顯微鏡之觀察 34
5-2-2 γ-聚麩胺酸/膠原蛋白生物組織黏著劑之最適組成比例 34
5-2-3 交聯劑(EDC)濃度對於組織黏著劑黏滯性質的影響 34
5-2-4 膠原蛋白濃度對組織黏著劑黏滯性質的影響 35
5-2-5 γ-聚麩胺酸濃度對組織黏著劑黏滯性質的影響 35
5-2-6 γ-聚麩胺酸/膠原蛋白生物組織黏著劑之拉伸強度測試 35

5-3 γ-聚麩胺酸/膠原蛋白組織黏著凝膠含水率測試 36
5-4 In vitro細胞毒性測試 36
5-5 In vivo動物實驗測試 37

第六章 結論 50

參考文獻 51
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