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研究生:黃婷郁
研究生(外文):Ting-Yu Huang
論文名稱:以仿生模板法製備高比表面積聚鄰甲氧基苯胺生物基材及其在神經幹細胞的行為研究之探討
論文名稱(外文):Preparation and Characterization of High Surface Area Biomimetic Poly(ortho-methoxyaniline) Substrate for Neural Stem Cells Behavior
指導教授:葉瑞銘葉瑞銘引用關係
指導教授(外文):Jui-Ming Yeh
學位類別:碩士
校院名稱:中原大學
系所名稱:化學研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:76
中文關鍵詞:聚鄰甲氧基苯胺仿生神經幹細胞
外文關鍵詞:Poly(ortho-methoxyaniline)BiomimeticNeural stem cell
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本實驗利用化學氧化聚合法聚合出聚鄰甲氧基苯胺 (poly (ortho-methoxyaniline),POMA),利用凝膠滲透層析 (GPC) 及傅利葉紅外線吸收光譜儀 (FT-IR) 鑑定重量平均分子量 ( ) 及結構。將 POMA 溶於N-甲基吡咯烷酮 (NMP) 溶劑中,利用聚二甲基矽氧烷 (PDMS) 複製紫柄千年芋葉面表面結構做為模板,製備出具有仿生微結構之 POMA 生物基材,其表面因具微米尺度的疣狀凸起結構以及奈米尺度的皺摺,而提升其高比表面積,更進一步利用場發射掃描式電子顯微鏡 (SEM) 及原子力顯微鏡 (AFM) 鑑定其表面型態。接著,更進一步針對神經再生修復進行探討,神經幹細胞培養在 具高比表面積POMA 生物基材上探討其貼附性及細胞存活率。透過貼附實驗利用 Trypan blue 計數發現神經幹細胞經過一天貼附後,在基材上具有貼附性;生物相容性及細胞存活分析實驗初步結果發現培養在基材上的神經幹細胞有停滯生長之現象,最後利用免疫螢光染色實驗證實具高比表面積 POMA 生物基材可促使神經幹細胞分化之能力。

In this study, we synthesized a polymer, poly(ortho-methoxyaniline) (POMA), by chemical oxidative polymerization. The weight-average molecular weight ( ) and structure were characterized by gel permeation chromatography (GPC) and Fourier transform infrared spectroscopy (FT-IR), respectively. POMA was dissolved in N-methyl-2-pyrrolidone (NMP) and the biomimetic POMA substrate was fabricated by using a transparent polydimethylsiloxane (PDMS) as template with negative patterns of a Xanthosoma sagittifolium leaf. The multiscale papilla-like and nanoscale texture increase the specific surface area. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) were used to determine the morphology of as-prepared POMA. Subsequently, studies on cell–substrate interactions were carried out by culturing neural stem cells (NSCs) on the high surface area biomimetic POMA substrate and assessing their adhesion and cell viability. Results of cell adhesion assay, biocompatibility and viability assay confirmed that biomimetic POMA substrate showed better NSCs attachment but induced growth arrest. The immunofluorescence staining Taken together, our data suggest that biomimetic POMA substrate could enhance the efficiency of NSCs differentiation.

中文摘要 I
Abstract II
致謝 III
目錄 IV
圖表目錄 VIII
第一章緒論 1
1-1前言 1
1-2理論基礎 2
1-2-1生醫材料 2
1-2-1-1生醫材料簡介 2
1-2-1-2生醫材料種類 3
1-2-1-3高分子生醫材料於組織工程之應用 5
1-2-2 聚苯胺 6
1-2-2-1 聚苯胺之簡介 6
1-2-2-2 聚苯胺之合成 9
1-2-2-3 聚苯胺之穩定性 10
1-2-2-4 聚苯胺之衍生物 11
1-2-2-5聚苯胺及其衍生物於組織工程之應用 12
1-2-3 仿生 13
1-2-3-1何謂仿生 13
1-2-3-2仿生之應用 13
1-2-4神經幹細胞 16
1-2-4-1神經幹細胞之簡介 16
1-2-4-2 神經新生 17
1-3 文獻回顧 18
1-4 研究動機與目的 26
第二章實驗 27
2-1 實驗藥品 27
2-2 實驗儀器 31
2-3 聚鄰甲氧基苯胺生物基材之製備 35
2-3-1 合成聚鄰甲氧基苯胺 35
2-3-2 PDMS模板製備 35
2-3-3 平面結構 POMA 生物基材製備 36
2-3-4 具仿生微結構 POMA 生物基材 36
2-4神經幹細胞之培養 37
2-4-1 神經幹細胞取得與繼代 37
2-4-2 細胞培養 38
2-4-3 細胞貼附性 38
2-4-4 生物相容性測試 38
2-4-5 細胞存活分析 39
2-4-6 免疫螢光染色 41
2-4-7 高溫高壓滅菌 (Autoclave) 42
第三章結果與討論 43
3-1 聚鄰甲氧基苯胺之分子量鑑定 43
3-2 聚鄰甲氧基苯胺之結構鑑定 43
3-3 聚鄰甲氧基苯胺生物基材之表面結構鑑定 45
3-3-1 掃描式電子顯微鏡 (SEM) 45
3-3-1-1 千年芋葉片及 PDMS 模板 45
3-3-1-2 平面結構、仿生結構聚鄰甲氧基苯胺生物基材 46
3-3-1-3 滅菌後之仿生結構聚鄰甲氧基苯胺生物基材 49
3-3-2原子力顯微鏡 (AFM) 49
3-4 聚鄰甲氧基苯胺生物基材之熱性質鑑定 51
3-5神經幹細胞於聚鄰甲氧基苯胺生物基材培養之應用探討 53
3-4-1 神經幹細胞在聚鄰甲氧基苯胺基材之貼附測試 53
3-4-2 神經幹細胞在聚鄰甲氧基苯胺基材之生物相容性測試 54
3-4-3 神經幹細胞在聚鄰甲氧基苯胺基材之細胞存活分析 56
3-4-4神經幹細胞在聚鄰甲氧基苯胺基材之免疫螢光染色 57
第四章結論及未來展望 62
第五章 參考文獻 63

圖表目錄
表 1-1 生物醫學用上常被使用的高分子...............................................4
圖 1-1 苯胺八聚體之五種不同鹼化型式的氧化態...............................7
圖 1-3 自然界中仿生的對象及其功能之概觀.....................................14
圖 1-4 自然界中仿生之範例.................................................................15
圖 1-5 PDMS 生物基材..........................................................................19
圖 1-6 人類胚胎幹細胞於 PDMS 生物基材之基因表現定量 .........19
圖 1-7 (A)老鼠神經始祖細胞之培養條件及(B)PDMS 生物基材 .....20
圖 1-8 老鼠神經始祖細胞於生物基材上分化後之 TH 及 PITX3 基
因表現量...................................................................................................21
圖 1-9 生物基材上之神經突長長度及細胞沿基材生長之比例.........21
圖 1-10 利用電紡絲技術所製備出之 PCL 纖維 SEM 圖...............22
圖 1- 11 老鼠神經幹細胞在 PCL 纖維基材之增生..........................22
圖 1-12 以電漿法製備高比表面積基材...............................................23
圖 1-13 人類胚胎幹細胞於電漿改值後之基材的增生實驗...............24
圖 1-14 快速模板製備示意圖...............................................................24
圖 1-15 具微米、奈米結構之粗糙表面基材 SEM 圖與成骨細胞貼附
於基材之貼附率 ......................................................................................25
IX
圖 2-1 PDMS 模板製備流程圖..............................................................36
圖 2-2 活 細 胞 染 劑 (Calcein AM) 及 死 細 胞 染 劑 (Ethidium
homodimer-1,EthD-1) 於細胞中作用機制 .......................................40
表 3-1 為 POMA 合成之分子量與分子量分布情形.........................43
圖 3-1 聚鄰甲氧基苯胺之傅利葉轉換紅外線光譜圖譜.....................44
圖 3-2 千年芋巨觀及維觀下之圖像.....................................................45
圖 3-3 POMA 生物基材.........................................................................47
圖 3-4 SEM 下之 POMA 生物基材 ....................................................48
圖 3-5 (a)滅菌前、(b)滅菌後之仿生結構 POMA 生物基材之 SEM
俯視圖.......................................................................................................49
圖 3-6 (a)平面結構、(b)具仿生微結構 POMA 生物基材之 AFM 圖
...................................................................................................................50
圖 3-7 聚鄰甲氧基苯胺之熱重分析圖.................................................52
圖 3-8 神經幹細胞於生物基材之貼附測試..........................................54
圖 3-9 神經幹細胞於生物基材之增生測試.........................................55
圖 3-10 神經幹細胞於生物基材之存活分析.......................................57
圖 3-11 神經幹細胞於生物基材之免疫螢光染色...............................60

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