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研究生:潘勁屹
研究生(外文):Chin-i Pan
論文名稱:聚己內酯表面活化與表面奈米化對細胞生長之研究
論文名稱(外文):Enhancing cell growth on poly(ε-caprolactone) surface by RGD modification, and chitosan with nanometer roughness
指導教授:鍾次文
指導教授(外文):Tze-wen Chung
學位類別:碩士
校院名稱:國立雲林科技大學
系所名稱:工業化學與災害防治研究所
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:2004
畢業學年度:92
語文別:中文
論文頁數:85
中文關鍵詞:奈米表面聚己內酯DSPE-PEGRGD纖維母細胞內皮細胞
外文關鍵詞:fibroblastHUVECRGDDSPE-PEGPCLnano-surface
相關次數:
  • 被引用被引用:10
  • 點閱點閱:146
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
本研究以RGD peptide和chitosan表面奈米化兩種方法對聚己內酯(poly(ε- caprolactone))進行表面修飾,以促進細胞於材料表面的貼附與生長。
方法一: 以溶劑對PCL表面進行輕微侵蝕,並以DSPE- PEG鑲於PCL表面(PCL-PEG),並用光化學反應使RGD接枝於PCL-PEG表面(PCL-PEG-RGD) ; 經原子力顯微鏡(AFM)量測,各材料皆為平滑的表面,Ra值皆在40 nm以下,且混合溶劑的侵蝕只對材料表面作輕微修飾,並不會對材料本身結構有太大的改變,並能增加材料表面的生物活性; 由細胞螢光染色圖顯示,內皮細胞(HUVEC)於PCL-PEG-RGD表面貼附生長的情況最佳,同時,其結果與細胞活性測試(MTT test)相符合,PCL和PCL-PEG並不適合生長。
方法二: 利用丙酮於PCL表面進行侵蝕,作為chitosan奈米表面拓印之用;並將PCL表面接枝具奈米表面之幾丁聚醣(chitosan)薄膜。纖維母細胞(fibroblast)貼附生長實驗顯示,具奈米表面的PCL- chitosan材料有最佳的貼附效果,PCL-chitosan平面材料次之,PCL較差;如:細胞於polystyrene 培養皿之成長為100%,經由七天之培養,fibroblast成長後之MTT測試,具奈米表面之PCL-chitosan,平面之PCL-chitosan及PCL表面分別61.7%,37.6%,17.6%。
poly(ε- caprolactone)表面利用RGD peptide和chitosan 表面奈米化等方法活化確實會促進細胞的貼附與生長,未來具組織工程應用價值。
This study provides two techniques to modify PCL surface to enhance the adhesion and growth of cells on the surfaces.
Embedded DSPE-PEG to PCL surface by solvent etching-embedded technique, following with RGD grafting by photochemical reactions. The results show that the solvent etching-embedded techniques only caused nano-meter surface disturbance (< 40nm) for all of the modified surfaces. In addition, the HUVEC cells are well growth on the PCL-PEG-RGD surface by the observation of microscopy that demonstrates the effectiveness of the techniques.
Nano-meter roughness of chitosan surface(CS), re-produced by solvent-etched PCL surface, was grafted to PCL plain surface to active PCL for the growth of fibroblast. The growth of fibroblast on nanometer roughness PCL-CS surface well than plain PCL-CS surface and much better than PCL surface. For example, Based on polystyrene cell culture well as 100% for cell growth for 7 days. The MTT tests for fibroblast are 61.7%, 37.6% and 17.6% for nanometer roughness of PCL-CS, plain surface of PCL-CS and PCL surface, respectively. Through the study both techniques for PCL surface modifies can effectively enhance the growth of cell and can be applied to tissue engineering.
目錄
中文摘要........................................I
英文摘要......................................III
謝誌...........................................IV
目錄............................................V
圖索引.........................................IX
表索引.........................................XI


第一章、緒論....................................1
§1-1生醫材料回顧................................1
§1-1-1聚己內酯(poly(ε-caprolactone))...........2
§1-1-2幾丁聚醣.................................4
§1-2生醫材料與細胞貼附..........................7
§1-3細胞活化因子修飾生醫材料...................11
§1-4天然交聯劑-綠槴子素(genipin) ..............13
§1-5奈米表面對細胞貼附/生長的影響..............15
§1-6實驗動機...................................17


第二章、實驗設備與方法.........................18
§2-1 實驗藥品..................................18
§2-2 實驗設備..................................19
§2-3 實驗流程..................................20
§2-4 實驗方法與步驟............................22
§2-4-1 材料製備(一): PCL-PEG-RGD製備..........22
§2-4-2 材料製備(二): PCL-chitosn合成與
chitosan奈米表面...............................25
§2-5 PCL-PEG-RGD與PCL-chitosn(奈米表面)分析....26
§2-5-1(a) 掃瞄式電子顯微鏡(SEM)...............26
§2-5-1(b) 原子力顯微鏡(AFM)...................26
§2-5-2 接觸角量測 (Contact angle measurement)...................................26
§2-5-3減弱全反射-傅立葉轉換紅外線光譜儀
(ATR-FTIR).....................................26
§2-5-4 元素分析儀(Element Analysis)...........27
§2-6細胞於材料上貼附生長之情形、細胞活性測試- MTT Test.......................................28
§2-6-1細胞培養................................28
§2-6-2細胞於材料上貼附生長之情形..............29
§2-6-3 細胞活性測試—MTT Test.................29


第三章、結果與討論(一):PCL-PEG-RGD.............30
§3-1 PCL-PEG-RGD之材料表面型態分析.............30
§3-1(a) 掃瞄式電子顯微鏡(SEM).................30
§3-1(b) 原子力顯微鏡(AFM).....................30
§3-2 PCL-DSPE-PEG-GRGD之材料表面特性析.........34
§3-2-1 接觸角量測 (Contact angle measurement.34
§3-2-2傅立葉轉換紅外線光譜儀(ATR-FTIR)........36
§3-2-3元素分析儀(Element Analysis)............38
§3-3 內皮細胞細胞於材料上之貼附與生長..........39
§3-3(a) 細胞貼附與生長........................39
§3-3(b) 細胞活性測試—MTT Test................42

第四章、結果與討論(二):PCL-chitosan與chitosan表面奈米化
§4-1 PCL-chitosan材料表面型態分析..............45
§4-1(a) 掃瞄式電子顯微鏡(SEM).................45
§4-1(b) 原子力顯微鏡(AFM).....................45
§4-2 PCL-chitosan材料穩定性的探討..............49
§4-2(a) 膨潤度對材料穩定性之影響..............49
§4-2(b) Chitosan release對材料穩定性的影響....49
§4-3 傅立葉轉換紅外線光譜儀(ATR-FTIR) .........52
§4-4 纖維母細胞於PCL-chitosan材料上之貼附與生長54
§4-4-1細胞貼附與生長.........................54
§4-4-2 細胞於不同數天培養後之細胞活性比較....57

第五章、結論...................................61

未來展望.......................................63
參考文獻.......................................64


圖索引
圖 1-1 Poly(ε-caprlactone)之分子式..............3
圖 1-2 chitosan之分子結構式.....................5
圖 1-3 細胞貼附於材料之機制....................11
圖 1-4 Genipin之分子結構式.....................14
圖 2-1 PCL-PEG-RGD製備流圖.....................20
圖 2-2 PCL-chitosan(nm)表面製備流程圖..........21
圖 2-3 GRGD之分子結構式........................23
圖2-4 GRGD與SANPAH反應示意圖...................24
圖2-5 SANPAH-GRGD光照接枝於PCL-PEG表面.........24
圖2-6 chitosan表面接上HMDI示意圖...............25
圖2-7 chitosan-HMDI接枝PCL示意圖...............25
圖3-1 電子顯微鏡拍攝之材料表面.................32
圖3-2 原子力顯微鏡拍攝之材料表面型態...........33
圖3-3 各種不同材料之接觸角.....................35
圖3-4 ATR-FTIR分析圖..........................37
圖3-5 DSPE-PEG結構式..........................38
圖3-6 內皮細胞於材料生長36小時之情形...........41
圖3-7 細胞於各種材料上之MTT量測比較............44
圖4-1 電子顯微鏡拍攝之材料表面.................47
圖4-2 原子力顯微鏡拍攝之材料表面型態...........48
圖4-3 PCL-chitosan之膨潤度.....................51
圖4-4 PCL-chitosan之穩定度.....................51
圖4-5 ATR-FTIR分析圖...........................53
圖4-6 纖維母細胞於材料生長72小時之情形.........56
圖4-7 纖維母細胞於材料之MTT量測比較............60
圖4-8 纖維母細胞於材料之MTT量測比較............60



表索引
表1-1 細胞膜上得黏著接受器(adhesion receptors).10
表3-1 細胞於各種材料上之生長速率...............43
表4-1 細胞於各種材料上培養三天之生長速率.......59
表4-2 細胞於各種材料上培養七天之生長速率.......59
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