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研究生:洪伊昇
研究生(外文):Yi-Sheng Hung
論文名稱:膠原蛋白/玻尿酸奈米纖維基質之製備及其傷口癒合之應用
論文名稱(外文):Preparation of collagen/Hyaluronic acid nanofibrous matrix and its applications in wound healing
指導教授:許富銀
指導教授(外文):Fu-Yin Hsu
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:93
中文關鍵詞:膠原蛋白玻尿酸電氣紡絲
外文關鍵詞:collagenhyluronic acidelectrospinning
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  • 被引用被引用:3
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  • 下載下載:289
  • 收藏至我的研究室書目清單書目收藏:1
電氣紡絲之系統廣泛被應用於組織工程上,主要之原因為其操作過程簡單並且可有效率的製造出奈米等級之纖維;其纖維之構形提供較大的表面積,以利細胞貼附生長。於電氣紡絲系統中,膠原蛋白/玻尿酸之溶解度及電氣紡絲不同參數(如:電壓、不同溶劑比、不同重量百分比)會對纖維直徑之影響。本實驗以formic acid / HFIP混合溶液作為電氣紡絲之最佳溶劑,由掃描式電子顯微鏡結果圖得知其電壓、不同溶劑比及不同重量百分比之參數對膠原蛋白/玻尿酸奈米複合纖維之直徑大小沒有顯著的差異。而由螢光顯微鏡及FT-IR結果圖可證明膠原蛋白與玻尿酸的確存在於膠原蛋白/玻尿酸共紡之複合奈米纖維內。於細胞的結果圖中,可以得知膠原蛋白/玻尿酸重量百分比為16/3於第二天及第四天時,細胞生長的情形與培養皿有明顯的差異(P<0.05),而在第六天時,彼此間沒有明顯的差異(P>0.05),且細胞型態以round-like呈現也和培養皿的spindle-like型態有所差異。基因表現的結果圖中,可以發現膠原蛋白/玻尿酸重量百分比為16/3的複合奈米纖維,可使MMP-1/TIMP-1比值增加,由此可推知膠原蛋白/玻尿酸重量百分比為16/3之基材可能會減少疤痕組織之形成。由以上之結果可得知本實驗所開發的膠原蛋白/玻尿酸複合奈米纖維於傷口癒合的應用上,極具有開發之價值。
Electrospinning is a simple and effective method for producing nanofibers. The fibrous matrix had high specific surface area. This property could enhance the cell adhesion and proliferation. We successfully fabricated Type I collagen / hyaluronic acid nanofibrous matrix in HFIP/ formic acid system. Scanning electron microscope revealed that different factors, including voltage, concentration, and solution ratio, were not significantly effected the average diameter of type I collagen / hyaluronic acid nanofiber. On the other hand, we utilized FTIR and fluorescent label method to prove the nanofibers containing hyaluronic acid and collagen. Furthermore, foreskin cells cultured on the various weight ratio of collagen/hyaluronic acid nanofibrous matrix displayed differential cell attachment, proliferation rate, cell morphology and gene expression. Cells expressed higher levels of MMP-1/TIMP-1 ratio on the ratio of collagen to hyaluronic acid was 16/3. The increased ratio of MMP to TIMP expression, which is characteristic of scales wound. Together the data demonstrated that collagen/hyaluronic acid nanofibrous matrix has potential as a wound dressing for skin regeneration.
目錄
中文摘要 I
Abstract II
目錄 III
圖目錄 VI
表目錄 VIII
表目錄 VIII
第一章 前言 1
第二章 研究動機 3
第三章 文獻回顧 5
3.1 皮膚 5
3.2 傷口癒合過程 7
3.3 傷口治療之方式 9
3.4 人工皮膚具備之條件 11
3.5 市售之人工皮膚 13
3.6 基質之製備 16
3.7 電氣紡絲 17
3.8 膠原蛋白 21
第四章 實驗藥品 29
第五章 實驗儀器與耗材 31
第六章 實驗方法 33
6.1 膠原蛋白純化 33
6.2 膠原蛋白之定性及定量 35
6.3 膠原蛋白、玻尿酸及膠原蛋白/玻尿酸複合物之溶解度測試 38
6.4 不同重量百分比之膠原蛋白/玻尿酸混合溶液配置 39
6.5 膠原蛋白/玻尿酸複合奈米纖維製成 40
6.6 膠原蛋白螢光之標定 41
6.7 玻尿酸螢光之標定 42
6.8 不同重量百分比之膠原蛋白/玻尿酸複合奈米纖維之1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)交鏈 43
6.9 MTT assay 46
6.10 不同重量百分比之膠原蛋白/玻尿酸複合奈米纖維基材對纖維母細胞之影響 47
6.11 基因表現 49
第七章 研究結果與討論 53
7.1 膠原蛋白/玻尿酸之溶劑選擇 53
7.2 膠原蛋白/玻尿酸奈米複合纖維之製備 55
7.3 EDC交鏈時間對交鏈膠原蛋白/玻尿酸之影響 61
7.4 Rhodamine-膠原蛋白/FITC-玻尿酸螢光標定之分析 62
7.5 膠原蛋白、玻尿酸及膠原蛋白/玻尿酸之FT-IR成分分析 64
7.6 不同重量百分比之膠原蛋白/玻尿酸複合奈米纖維基材對纖維母細胞生長之影響 68
7.7 不同重量百分比之膠原蛋白/玻尿酸複合奈米纖維基材對纖維母細胞型態之影響 71
第八章 結論 76
第九章 參考文獻 77

圖目錄
圖一:皮膚構造 6
圖二:真皮層構造及組成分子 6
圖三:電紡之裝置圖 20
圖四:膠原蛋白D-period之構形 25
圖五: 玻尿酸之結構 28
圖六:EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride)之構 44
圖七:膠原蛋白與玻尿酸間之交鏈示意圖 45
圖八:不同重量百分比之膠原蛋白/玻尿酸之混合溶液於電壓23KV、流速為0. 51c.c. /hr、工作距離為10 cm、針頭為18G之電紡條件下,膠原蛋白/玻尿酸之複合奈米纖維於電子掃描顯微鏡圖 57
圖九:不同溶劑比之膠原蛋白/玻尿酸之混合溶液於電壓23KV、流速為0. 51c.c. /hr、工作距離為10 cm、針頭為18G之電紡條件下,膠原蛋白/玻尿酸之複合奈米纖維於電子掃描顯微鏡圖 58
圖十:膠原蛋白/玻尿酸之HFIP/formic acid溶劑比為7/3混合溶液於流速為0. 51c.c./hr、工作距離為10 cm、針頭為18G、不同電壓之電紡條件下,膠原蛋白/玻尿酸之複合奈米纖維於電子掃描顯微鏡圖 59
圖十一:膠原蛋白/玻尿酸奈米複合纖維,利用EDC進行交鏈,於不同時間作用後,在電子掃描顯微鏡下觀察所得之型態 61
圖十二:rhodamine-膠原蛋白/FITC-玻尿酸溶液經電紡所得之奈米複合纖維於螢光顯微鏡下之結果圖 63
圖十三:膠原蛋白、玻尿酸及其混合物於FTIR之結果圖 66
圖十四:纖維母細胞培養於不同基材下,培養二天之MTT assay比較圖 69
圖十五:纖維母細胞培養於不同基材下,培養四天之MTT assay比較圖 69
圖十六:纖維母細胞培養於不同基材下,培養六天之MTT assay比較圖 70
圖十七:纖維母細胞培養在不同基材(培養皿、膠原蛋白纖維及膠原蛋白/玻尿酸複合奈米纖維 72
圖十八:不同重量比之膠原蛋白/玻尿酸奈米纖維對foreskin cell之β-acitin、collagen-1、MMP-1及TIMP-1基因表現的影響 75

表目錄
表1 市售之人工皮膚 15
表2: 組織器官內之膠原蛋白之類型 23
表3: 膠原蛋白應用之優點 24
表4:SDS-PAGE各標準溶液配置 37
表5:β-actin、Collagen、MMP-1、TIMP-1 引子序列之設計 52
表6:不同溶劑對玻尿酸和膠原蛋白及其混合物之溶解度 54
表7:膠原蛋白/玻尿酸物理特性結果表 60
表8:膠原蛋白及玻尿酸於FT-IR之特徵波長 67
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