許多蛋白質會發生異常摺疊，形成非原生態的結構，進而形成蛋白質聚集物。現今研究發現有將近20種蛋白質會導致特定的疾病，但是由於尚未找出有效預防以及治癒的方式，所以依舊困擾許多病患以及研究者。而這些致病蛋白質的氨基酸序列、分子大小、以及折疊的三維結構不盡相同，但是一但形成錯誤折疊，最終都是形成不可溶的類澱粉纖維。許多研究發現這些蛋白質聚集物的生成會受到環境的影響，像是pH值、溫度、或是添加共溶劑像是酒精、鹽類、以及金屬離子等等。所以在本實驗中，試著改變溶劑環境、pH值以及添加短胜肽和樹枝狀高分子來孵育人類胰島素，藉由紅外光譜、圓二色分光光譜、螢光光譜以及原子力顯微鏡等來觀察二級結構和表面形貌的變化。結果顯示當提高醇類百分比或是增加醇類碳鏈長，都會導致CD光譜產生變化。在纖維表面型態的觀察中發現，提高醇類百分比或是增加醇類碳鏈長都會抑制胰島素纖維的形成，這是可能是來自於疏水基團外翻以及溶劑分子影響。而降低pH值會促使結構加速由α-helix 轉變為 β-sheet，因此更易形成大型聚集物。這結果可能來自於鹽酸水溶液中之氯離子會覆蓋胰島素纖維上的電荷，削弱纖維間的電荷斥力而導致聚集。

Many proteins can misfold to form non-native structures and induce aggregation. Previous studies had found that more than 20 kinds of proteins may cause specific diseases that under ineffective prevention and treatment, and greatly perplexed many patients and researchers. Either the amino acid sequences, or molecular size, or the folded three-dimensional conformation of these pathogenic proteins is quite different, all of them will lead to the formation of insoluble amyloid fibers after misfolding. Herein, we investigated the environmental changes by the alcohol content、pH value and adding short peptides and dendrimers in solvent incubated with human insulin. We used circular dichroism (CD) spectroscopy、fourier transform infrared spectroscopy (FT-IR) and fluoresce spectroscopy to obtain the structural transition of the insulin, and gain the morphology information of fibril by atomic force microscopy (AFM). The results showed that the increasing percentage of alcohol or increasing carbon chain length of alcohols can cause a difference CD spectra. The surface morphology of insulin fibers by AFM imaging indicated that either increasing the percentage of alcohol or increasing alcohol carbon chain length will inhibit insulin fiber formation. This phenomenon was attributing to hydrophobic group expose to solution and solvent molecular may prevent nucleus to contact. And lowering the pH will undergo conformational change from α-helix to β-sheet form that causes the formation of amyloid plaques. This result may be attributed to Cl- that covered the surface charge of insulin fibril, then weaken the original electrostatic repulsion among insulin fibrils and result in their aggregation.

論文審定書+i
中文摘要+ii
Abstract+iii
目錄+iv
圖次+vii
表次+x
第壹章、 緒論+1
1-1 前言+1
1-2 研究背景+2
1-3 研究動機+4
第貳章、 儀器與實驗+6
2-1 圓二色光譜儀 (Circular Dichroism, CD)+6
2-1-1 簡介+6
2-1-2 蛋白質的圓二色光譜+9
2-1-3 參數設定+10
2-2 傅立葉轉換紅外線光譜儀 (Fourier transform infrared spectroscopy)+11
2-2-1 原理+11
2-2-2 蛋白質的紅外光譜+14
2-2-3 參數設定+15
2-3 原子力顯微鏡(Atomic force microscopy, AFM)+16
2-3-1 簡介+16
2-3-2 原理+16
2-3-3 工作模式+18
2-3-4 使用儀器及參數設定+20
2-4 界達電位粒徑分析儀(Zeta potential analyzer)+21
2-4-1 簡介+21
2-4-2 原理+23
2-4-3 儀器+23
2-5 螢光光譜儀(Fluorescence Spectrometers)+24
2-5-1 簡介+24
2-5-2 實驗儀器與參數設定+26
2-6 實驗藥品與材料+27
2-6-1 實驗藥品+27
2-6-2 實驗材料+28
2-7 樣品製備+28
2-7-1 基板準備+28
2-7-2 胰島素纖維溶液製備+28
2-7-3 胰島素纖維與胜肽和樹枝狀聚合物共生長之溶液配置+29
2-7-4 CD樣品製備+30
2-7-5 IR樣品製備+30
2-7-6 界達電位樣品製備+30
2-7-7 ThT分析樣品製備+30
第參章、 結果與討論+31
3-1 人類胰島素於醇類環境中孵育的影響+31
3-1-1 前言+31
3-1-2 人類胰島素於乙醇水溶液中孵育的CD及FT-IR光譜變化+33
3-1-3 藉由AFM觀察人類胰島素於乙醇水溶液中孵育之形貌變化+34
3-1-4 人類胰島素於醇類水溶液中孵育的CD及FT-IR光譜變化+35
3-1-5 藉由AFM觀察人類胰島素於醇類水溶液中孵育之形貌變化+36
3-1-6 結果討論+37
3-1-7 結論+43
3-2 不同pH值孵育環境對人類胰島素成長的影響+44
3-2-1 前言+44
3-2-2 人類胰島素於不同pH值中的CD及FT-IR光譜變化+45
3-2-3 藉由AFM觀察人類胰島素於不同pH值中孵育之形貌變化+47
3-2-4 ThT分析+48
3-2-5 結果討論+50
3-2-6 結論+53
3-3 胜肽對人類胰島素成長的影響+55
3-3-1 前言+55
3-3-2 實驗結果+56
3-3-3 討論+60
3-3-4 結論+65
3-4 樹枝狀分子對人類胰島素纖維化的抑制+66
3-4-1 前言+66
3-4-2 實驗結果+67
3-4-3 討論+69
3-4-4 結論+71
第肆章、 結論+72
第伍章、 參考文獻+74

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