博碩士論文 92324031 詳細資訊




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姓名 邱秀玫(Hsiu-Mei Chiu)  查詢紙本館藏   畢業系所 化學工程與材料工程學系
論文名稱 利用表面電漿共振儀及恆溫滴定微卡計探討微脂粒與類澱粉胜肽交互作用之動力學及熱力學研究
(Kinetics and Thermodynamics of Liposome & ß-amyloid Interactions by Surface Plasmon Resonance and Isothermal Titration Microcalorimetry)
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摘要(中) 類澱粉胜肽(ß-amyloid,Aß)被認為是引起阿茲海默症的主要原因。Aß是具有自我聚集能力的兩性胜肽,一但從單體聚集至纖維狀(fibril)的結構就會毒殺神經細胞,但到目前為止Aß是如何引發細胞毒性及疾病的作用機制仍然不清楚,所以本研究主要是探討Aß與微脂粒之交互作用,以不同培養時間的Aß與不同組成之微脂粒(DPPC、DPPG添加GM1及膽固醇)進行討論,以期能說明Aß與細胞膜之作用行為。整個研究的主題分為兩個部份,第一部份是利用圓二色光譜儀(CD)、螢光光譜儀(Fluorescence spectroscopy)及原子力顯微鏡(AFM)偵測Aß隨培養時間其結構的變化;第二部份是利用表面電漿共振儀(SPR)及恆溫滴定微卡計(ITC)探討Aß與微脂粒交互作用之動力學及熱力學。
在Aß結構的偵測方面,培養ㄧ天後於AFM的結果可看到纖維狀的形成,螢光及CD的結果也都可發現相同的現象。在Aß與微脂粒交互作用之動力學研究方面,培養零天的Aß與不同組成微脂粒作用其親和力大小為DPPG>DPPC/DPPG(75:25 molar ratio)>DPPC/GM1/膽固醇(5:3:2 molar ratio)>DPPG/20 mole%膽固醇>DPPC。由親和力強度可判斷培養零天的Aß與不同組成微脂粒交互作用主要是靠靜電作用力。並可發現Aß對於GM1有較高的親和力;膽固醇的添加會降低微脂粒的流動性而改變了Aß與微脂粒的交互作用,特別於吸附量與吸附後膜結構之維持。培養ㄧ天的Aß與微脂粒作用其親和力大小為DPPC>DPPG/20 mole%膽固醇>DPPC/DPPG(75:25 molar ratio)>DPPG>DPPC/GM1/cholesterol(5:3:2 molar ratio)。由親和力強度可判斷培養一天的Aß與不同組成微脂粒交互作用主要是靠疏水作用力。以上結果初步地說明了Aß與細胞膜之作用機制與Aß培養時間不同而有所改變。在Aß與微脂粒交互作用之熱力學研究方面,由熱量變化可以發現靜電作用力及疏水作用力會同時存在。
摘要(英) ß-amyloid(Aß)was believed to cause the prime factor of Alzheimer’s disease. However, the mechanism of the cytotoxicity and the disease caused by Aß is still unclear. The goal of this work is to study the interactions of various incubated time of Aß with designed liposomes. The objective of this investigation was achived by the following studies : The structural and aggrergation information of Aß by by circular dichroism spectroscopy(CD), by Thioflavin T fluorescence assay and monitored by atomic force microscopy(AFM). Futhermore, this investigation utilized surface plasmon resonance (SPR) and isothermal titration microcalorimetry (ITC) to measure the kinetics and binding enthalpy of Aß with the liposomes.
Kinetics of interactions of Aß and liposomes by SPR reveal the driving force of fresh Aß interacts with various liposomes is electrostatics. And fresh Aß have high affinity with GM1. Addition of cholesterol to the liposome could alter membrane fluidity and affect the interactions of fresh Aß with liposomes especially in the amount of absorbed Aß and maintained the structure of liposome after adsorbing. The driving force of the 1 day of incubation of Aß interacts with various liposomes is hydrophobicity. The binding enthalpy measurements between Aß and liposomes by ITC are endothermic reaction. When the composition of liposome is zwitterionic lipids, the interaction of Aß with liposomes is predominantly hydrophobic force; in contrast with the drive force of interaction of charged lipid with Aß is electrostatic force.
關鍵字(中) ★ 動力學
★ 表面電漿共振儀
★ 類澱粉胜肽
★ 阿茲海默症
★ 微脂粒
★ 焓
關鍵字(英) ★ beta-amyloid
★ kinetics
★ surface plasmon resonance
★ enthalpy
★ liposome
★ Alzheimer's disease
論文目次 第一章 前言 1
第二章 文獻回顧 3
2.1 表面電漿共振 3
2.1.1 表面電漿現象原理 3
2.1.2 光學激發表面電漿之方式 4
2.1.3 SPR檢測生物反應 7
2.1.4 表面電漿共振光譜儀於分子交互作用上可提供之資訊 9
2.1.5 其他類型之表面電漿共振 10
2.1.5.1 長距離表面電漿共振 10
2.1.5.2 耦合電漿波導共振 11
2.1.5.3 表面電漿共振螢光光譜儀 12
2.1.5.4 金奈米粒子增強表面電漿光譜儀 13
2.2阿茲海默症與類澱粉胜肽 15
2.2.1 阿茲海默症(Alzheimer’s Disease,AD) 15
2.2.2 類澱粉胜肽(ß-amyloid,Aß) 19
2.2.3 類澱粉胜肽的結構 20
2.2.3.1類澱粉胜肽於溶液中的結構變化 22
2.2.3.2 生物細胞膜的存在與組成對Aß結構變化的影響 25
2.2.4 Aß與生物細胞膜的交互作用 30
2.2.4.1 Aß與生物細胞膜交互作用之動力學研究 30
2.2.4.2 Aß與生物細胞膜交互作用之熱力學研究 33
2.2.4.3 Aß與脂質單分子層的交互作用 33
2.2.4.4 Aß對細胞膜流動性的影響 34
2.2.5 目前阿茲海默症的治療方式 37
2.2.6 結論 39
第三章 實驗藥品與儀器設備 54
3.1 實驗藥品 54
3.2儀器設備 55
3.2.1表面電漿共振儀(Surface Plasmon Resonance) 56
3.2.1.1表面電漿共振儀之光學系統 56
3.3 實驗流程圖 60
3.4 實驗方法 61
3.4.1 PBS緩衝液的製備 61
3.4.2 微脂粒的製備 61
3.4.3 Aß溶液的製備 62
3.4.4 金膜表面改質 62
3.4.5 表面電漿共振儀之實驗 65
3.4.6 恆溫滴定微卡計實驗 66
3.4.7 圓二色光譜儀實驗 67
3.4.8 螢光光譜儀實驗 67
3.4.9 原子力顯微鏡之影像偵測實驗 68
第四章 結果與討論 69
4.1 Aß結構探討 69
4.1.1 CD實驗分析 69
4.1.2 Thioflavin T fluorescence實驗分析 74
4.1.3 AFM實驗分析 77
4.2 類澱粉胜肽(Aß)與微脂粒之交互作用 87
4.2.1 SPR實驗 87
4.2.1.1 金片表面改質 87
4.2.1.2不同狀態Aß(1-40)與不同微脂粒交互作用之動 力學探討 91
4.2.14 不同狀態Aß(1-40)與不同微脂粒交互作用之 AFM影像偵測 105
4.2.2 ITC實驗 115
4.2.2.1 Aß(25-35)與不同微脂粒之交互作用 115
4.2.2.2 Aß(1-40)與不同微脂粒交互作用 117
第五章 結論與建議 123
參考文獻 126
圖目錄
圖2.1衰逝全反射方式之色散關係曲線圖 5
圖2.2以ATR方式(Otto & Kretschmann configuration)耦合表面電漿波 6
圖2.3 SPR以角度測量的方式檢測生物反應 7
圖2.5 LRSPR之組態 10
圖2.6耦合電漿波導共振之構造與含有Rhodopsin之酯雙層(lipid bilayer),與G protein transducin發生交互作用之示意圖 11
圖2.7表面電漿共振螢光光譜儀之儀器架構裝置圖 12
圖2.8表面電漿共振螢光光譜儀-SPFS工作原理示意圖 13
圖2.9奈米粒子增強SPR之示意圖 14
圖2.10金奈米粒子強化SPR感測器之示意圖 14
圖2.11 阿茲海默症主要的病理特徵:老化斑塊與神經纖維糾結 16
圖2.12 類澱粉假說 18
圖2.13 Aß由類澱粉前驅蛋白(amyloid protein precursor ,APP) 經由ß-、g-分解酵素分解而形成 19
圖2.14 Aß形成纖維狀的模式- 成核聚合模式 21
圖2.15 Aß由單體形成纖維狀的過程 22
圖2.16 以GM1為介質,促進Aß形成纖維狀(fibril)的示意圖 27
圖2.17 glycospingolipids的結構 28
圖2.18螢光探針的化學結構與位於POPC脂單層膜的位置 36
圖2.19 Recruiting hypothesis 示意圖 41
圖3.1自組之表面電漿共振儀 58
圖3.2金膜改質步驟及在金膜上形成脂雙層膜之實驗流程示意圖….74
圖4.1使用圓二色光譜儀(CD)隨時間偵測20mM Aß(1-40)溶
於pH 7.4 PBS 在室溫下培養 71
圖4.2 為圖4.1結果整理,以固定波長197nm(隨意螺線結構) 及218nm (ß-摺疊板結構)表現20mM Aß(1-40)溶於 pH 7.4 PBS在室溫下培養其橢圓率變化之程度 71
圖4.3 為圖4.2中的ß-摺疊板結構的生成速率擬合曲線圖 72
圖4.4 使用圓二色光譜儀(CD)隨時間偵測20 mM Aß(25-35) 溶於pH 7.4 PBS 在室溫下培養 73
圖4.5 使用圓二色光譜儀(CD)隨時間偵測75mM Aß(25-35) 溶於pH 7.4 PBS 在室溫下培養 73
圖4.6 使用ThT fluorescence分析20mM Aß(1-40)溶於 pH 7.4 PBS在室溫下培養其聚集程度變化 75
圖4.7 使用ThT fluorescence分析20mM Aß(1-40)溶於 pH 7.4 PBS在室溫下培養其聚集程度變化 75
圖4.8 為圖4.7中的纖維狀之生成速率擬合曲線圖 76
圖4.9 使用AFM 偵測20mM Aß(1-40)溶於pH 7.4 PBS 在室溫 下培養1小時之影像圖 78
圖4.10 使用AFM 偵測20mM Aß(1-40)溶於pH 7.4 PBS 在室溫 下培養2小時之影像圖 79
圖4.11 使用AFM 偵測20mM Aß(1-40)溶於pH 7.4 PBS 在室溫 下培養3小時之影像圖 80
圖4.13 使用AFM 偵測20mM Aß(1-40)溶於pH 7.4 PBS 在室溫 下培養5小時之影像圖 82
圖4.14 使用AFM 偵測20mM Aß(1-40)溶於pH 7.4 PBS 在室溫 下培養6小時之影像圖 83
圖4.15 使用AFM 偵測20mM Aß(1-40)溶於pH 7.4 PBS 在室溫 下培養1天之影像圖 84
圖4.16 使用AFM 偵測20mM Aß(1-40)溶於pH 7.4 PBS 在室溫 下培養6天之影像圖 85
圖4.17 使用AFM 偵測20mM Aß(1-40)溶於pH 7.4 PBS 在室溫 下培養7天之影像圖 86
圖4.18 金片表面改質各階段之AFM俯視圖 89
圖4.19 金片表面改質各階段之AFM側視圖及3D圖 90
圖4.20 培養零天的20mM Aß(1-40)與不同微脂粒交互作用之 表面電漿共振感應圖 96
圖4.22 培養一天的20mM Aß(1-40)與不同微脂粒交互作用之 表面電漿共振感應圖 98
圖4.23 培養一天的20mM Aß(1-40)與不同微脂粒交互作用之 表面電漿共振感應圖 99
圖4.24 培養零天及一天的20mM Aß(1-40)與不同微脂粒交互作??用之表面電漿共振感應圖 100
圖4.25 培養零天的的20mM Aß(1-40)與DPPC製備的微脂粒交??互作用之影像圖 107
圖4.26 培養零天的的20mM Aß(1-40)與DPPG製備的微脂粒交??互作用之影像圖 107
圖4.27 培養零天的的20mM Aß(1-40)與DPPG/20mole%膽固醇??製備的微脂粒交互作用之影像圖 108
圖4.28 培養零天的的20mM Aß(1-40)與DPPC/GM1/cholesterol (5:3:2 molar ratio)製備的微脂粒交互作用之影像圖 108
圖4.29 培養零天的的20mM Aß(1-40)與DPPC/DPPG (75:25 molar ratio)製備的微脂粒交互作用之影像圖 109
圖4.30 培養一天的20mM Aß(1-40)與DPPC製備的微脂粒交互 作用之影像圖 110
圖4.31 培養一天的20mM Aß(1-40)與DPPG製備的微脂粒交互 作用之影像圖 110
圖4.32 培養一天的20mM Aß(1-40)與DPPG/20mole %膽固醇 製備的微脂粒交互作用之影像圖 111
圖4.33 培養一天的20mM Aß(1-40)與DPPC/GM1/膽固醇 (5:3:2 molar ratio)製備的微脂粒交互作用之影像圖 111
圖4.34 培養ㄧ天的20mM Aß(1-40)與DPPC/DPPG (75:25 molar ratio)製備的微脂粒交互作用之影像圖 112
圖4.35 培養零天及ㄧ天的20mM Aß(1-40)與不同微脂粒交互??作用之影像圖 113
圖4.36 培養零天及培養ㄧ天的20mM Aß(1-40)與不同微脂粒交 互作用之影像圖 114
圖4.37 Aß與微脂粒交互作用過程示意圖 120
圖4.38 Aß(25-35)與不同微脂粒交互作用之反應熱 121
圖4.39 Aß(1-40)與不同微脂粒交互作用之反應熱 121
圖4.40 Aß(25-35)與DPPC製備的微脂粒經ITC實驗後,
由CD偵測Aß(25-35)的結構變化 122
圖4.41 Aß(1-40)與DPPC添加20 mole%膽固醇製備的微脂粒
經ITC實驗後,由CD偵測Aß(1-40)的結構變化 122
表目錄
表2.1 表面電漿共振光譜儀可提供之資訊 9
表2.2 不同長度的Aß於不同溶液中其結構的變化 24
表2.3 影響Aß聚集的因子 29
表2.4 Aß對生物細胞膜流動性的影響 35
表2.5 類澱粉胜肽(Aß)的相關研究 42
表3.1 表面電漿共振儀使用元件及儀器一覽表 59
表4.1 使用AFM 偵測20mM Aß(1-40)其平均大小 83
表4.2 不同培養時間的Aß(1-40)與不同組成的微脂粒交互作用 之最大吸附量與脫附後殘留量 103
表4.3 使用Langmuir binding model 分析不同培養時間的 Aß(1-40)與不同組成的微脂粒交互作用的動力學參數 104
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指導教授 陳文逸(Wen-Yih Chen) 審核日期 2005-6-28
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