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研究生:陳興德
研究生(外文):Sing-De Chen
論文名稱:表面活化後之聚己內酯神經導管支架對類神經細胞生長之影響
論文名稱(外文):Enhancing cell growth on poly(ε-caprolactone) nerve conduit scaffold surface by chemical modification.
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:化學工程與材料工程研究所
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:2008
畢業學年度:96
語文別:中文
論文頁數:54
中文關鍵詞:幾丁聚醣聚己內酯RGDPC12神經生長因子tirofiban
外文關鍵詞:chitosanNGFPCLGRGDPC12tirofiban
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本研究將聚己內酯poly(ε- caprolactone)(PCL) scaffold以hybrid nerve growth factor(NGF)/GRGD、NGF/tirofiban進行表面改質,以促進其表面生物活性;再以類神經細胞,PC12為model cell,來探討改質後材料對PC12生長效果,希望可作為神經導管材料之應用。
製造PCL scaffold,再利用polyethylenimine(PEI)、chitosan(CS) 交聯於其上,再以光化學分別接枝NGF、GRGD、tirofiban、NGF/GRGD、NGF/tirofiban於PCL-CS;並以ATR-FTIR與ESCA及HPLC驗證接枝上分子(例如:ATR-FTIR在839 cm-1 1279 cm-1 1342 cm-1 分別證明材料上有GRD序列的特殊吸收峰,ESCA全掃描中證實有接枝tirofiban之材料皆可發現有硫元素(S)的存在,HPLC分析出GRGD與tirofiban之特殊吸收峰,且算出接枝率約為85%及87%。由細胞活性測試MTS顯示,接枝hybrid NGF/GRGD**>GRGD**>CS,接枝hybrid NGF/tirofiban*>tirofiban**>CS(**:P<0.05、*:P<0.1),且細胞DAPI染色結果與MTS測試結果相符合。
利用接枝hybrid NGF/GRGD、NGF/tirofiban為一方法,對類神經細胞PC12有促進貼附生長的效果,藉由NGF/GRGD、NGF/tirofiban改質聚酯類高分子作為一神經導管材料的確有其應用價值與潛力。
This study using hybrid nerve growth factor(NGF)/GRGD、NGF/tirofiban to modify PCL scaffold surface in order to enhance bioactivity;PC12 is a model cell used to discus the growth effect by chemical modification.
Prepare PCL scaffold,grafting chitosan by glutaradehye,then using SANPAH graft NGF、GRGD、tirofiban、NGF/GRGD、NGF/tirofiban.In ATR-FTIR, 839 cm-1 1279 cm-1 1342 cm-1 peaks demonstrate GRGD has grafted on the PCL surface. In ESCA, there are S atom appears, demonstrate tirofiban grafted on PCL surface. In HPLC, assay GRGD and tirofiban specific peaks,and the graft yield was 85% and 87%.The MTS assay,grafted hybrid NGF/GRGD**>GRGD**>CS ; hybrid NGF/tirofiban*>tirofiban**>CS(**:P<0.05、*:P<0.1), and the cell stain DAPI , shows the same results to MTS.
By using chemical modification, grafting NGF/GRGD、NGF/tirofiban,can enhance PC12 cell adhesion and growth. Through the study, grafting NGF/GRGD、NGF/tirofiban can effectively enhance the growth of cell and have good potential applied to nerve guide conduits.
中文摘要..................................................I
目錄.....................................................II
表目錄....................................................V
圖目錄...................................................VI


第一章、緒論…………………………………………………………....1
§1-1聚己內酯(poly(ε-caprolactone))……………………………………1
§1-2幾丁聚醣(chitosan)…………………………………………………3
§1-3 Arg-Gly-Asp序列…………………………………………………..5
§1-4神經生長因子(nerve growth factor)………………………………..7
§1-5 tirofiban……………………………………………………………..8
§1-6神經損傷與修復概述………………………………………………9
§1-7研究動機……...…………………………………………………...14

第二章、實驗設備與方法………………………………………………15
§2-1 實驗藥品………………………………………………………....15
§2-2 實驗設備…………………………………………………………16
§2-3 實驗流程…………………………………………………………17
§2-4 實驗方法與步驟……………………………………….……......18
§2-4-1 材料製備(一): PCL scaffold製備…………….……………..18
§2-4-2 材料製備(二): PCL-CS 合成………………………………..18
§2-4-3 材料製備(三): PCL-CS-NGF; PCL-CS-GRGD;
PCL-CS-GRGD/NGF; PCL-CS-tirofiban; PCL-CS-tirofiban/NGF
合成…………………………………………………………….20
§2-5 材料性質分析.........……...............................................................20
§2-5-1 掃瞄式電子顯微鏡(SEM)…………………………................20
§2-5-2減弱全反射-傅立葉轉換紅外線光譜儀(ATR-FTIR)……......20
§2-5-3電子光譜化學分析儀(ESCA)……………………..............20
§2-5-4高效能液相層析法 (HPLC)………………………………….21
§2-5-4-1高效能液相層析法:tirofiban
§2-5-4-2高效能液相層析法:GRGD
§2-6細胞於材料上貼附生長之情形、細胞活性測試- MTS Test..……22
§2-6-1 scaffold之細胞培養.………………………………….............22
§2-6-2細胞於材料上貼附生長之DAPI染色………………...........23
§2-6-3 細胞活性測試—MTS Test……….………………………….23
第三章、結果與討論………………………………………………….25
§3-1 PCL scaffold之材料表面型態………………………………….26
§3-1 掃瞄式電子顯微鏡(SEM)………….…………………………...26
§3-2 PCL-CS-GRGD/NGF; PCL-CS-tirofiban/NGF之材料表面特性分
析………………………………………………………………………26
§3-2-1傅立葉轉換紅外線光譜儀 (ATR-FTIR)…….……………….26
§3-2-2化學分析電子光譜儀 (ESCA)…………………….……….....30
§3-2-2-(a) ESCA全掃描分析………………………………………..30
§3-2-2-(b) ESCA區段掃描C1s分析…………………………………35
§3-2-3高效能液相層析 (HPLC)……………………………………...39
§3-3 PC12細胞於材料上之貼附與生長…………………………….....42
§3-3(a) 細胞貼附與生長—DAPI染色…………………………..…..42
§3-3(b) 細胞活性測試—MTS Test……..………………………..…..45
§3-3(c) 細胞貼附於支架上neurite生長情形………………………..46
第四章、結論……………………………………………………………48
參考文獻………………………………………………………………..50
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