臺灣博碩士論文加值系統

本研究主要是利用微脂粒（liposome）形成之脂雙層膜來建立一個模擬生物細胞膜系統，並且結合壓電石英晶體微天平（QCM）生物感測器以及恆溫滴定微卡計（ITC）等儀器，來研究探討生物分子與生物細胞膜間的交互作用機制。整個研究的主題分為兩個部分，第一個部分是探討微脂粒的組成及膽固醇含量對形成脂雙層膜的影響，第二個部分則是探討膽固醇的含量對蜂毒胜肽（melittin）與脂雙層膜的交互作用之影響。藉由壓電石英晶體微天平（QCM）及恆溫滴定微卡計（ITC）來探討蜂毒胜肽與脂雙層膜交互作用之動力學與熱力學，並利用原子力學顯微鏡（AFM）及雷射粒徑儀（DLS）偵測其構形的變化。
在形成脂雙層膜的部份，我們將金膜表面經過適當的化學改質修飾後，吸附上不同組成之微脂粒以形成脂雙層膜。實驗結果顯示，當微脂粒的主要脂質成分為卵磷脂（egg PC）時，微脂粒在吸附於金膜表面後，原本球體（Vesicle）的結構會破裂並形成平板狀的脂雙層膜（lipid bilayer）；而以二棕櫚酸磷脂醯膽鹼（DPPC）為主要脂質的微脂粒，在吸附於金膜表面後，其球形結構並無破裂現象而僅有些許變形。在蜂毒與脂雙層膜的交互作用之研究方面，實驗中使用二棕櫚酸磷脂醯膽鹼（DPPC）混和不同比例的膽固醇（0, 10, 30 %）作為微脂粒的成分，與不同濃度的蜂毒胜肽進行反應。由QCM、ITC、AFM、DLS等實驗顯示，蜂毒胜肽之穿膜行為是一熵驅動的程序，而熵的來源應為去水合之結果，並且用Two-state model來分析其動力學參數，發現當脂雙層膜的膽固醇含量高達30%時，明顯地抑制了蜂毒胜肽（melittin）的結合行為與穿膜行為。

Liposomes have been used to mimic natural cell membrane for various studies. In this investigation, the membrane models are adapted to study and establish a correlation between the penetration peptides upon binding with cell membranes and the biological specificities. Different structures of liposomes with different lipids and various content of cholesterol were observed on the biosensor (i.e. Quartz Crystal Microbalance (QCM)) with different liposome immobilization chemistry. With the mimic cell membrane system, the interaction kinetics and thermodynamics information of the melittin with the liposomes were studied by QCM, and isothermal titration calorimetry (ITC).The conformation of the liposomes was monitored by Atomic Force Microscope (AFM) on the sensor surface and by Dynamic Laser Scattering (DLS) in solution. The binding enthalpy measurements between peptides and the liposomes by ITC reveal that the penetration or insertion of melittin into the liposomes is entropy driven. The content of the cholesterol and the dehydration behavior of the presence of cholesterol in lipid bilayer contribute profoundly on the penetration behavior of the peptide.

中文摘要
英文摘要
目錄
圖目錄
表目錄
第一章 導論
第1-1節 研究動機與目的………………………………1
第1-2節 壓電石英晶體微天平…………………………3
1-2.1 生物感測器……………………………………………..3
1-2.2 壓電石英晶體於感測器上的應用……………………..4
1-2.3 氣相感測………………………………………………..7
1-2.4 液相感測………………………………………………..8
1-2.5 壓電石英晶體微天平的特性與感測原理……………14
第1-3節 生物細胞膜…………………………………..24
1-3.1 生物細胞膜的基本性質………………………………24
1-3.2 生物細胞膜的組成與結構……………………………25
1-3.3 常見磷脂質的種類與特性……………………………29
1-3.4 膽固醇(cholesterol) …………………………………32
1-3.5 細胞膜的流動性(fluidty) …………………………….35
第1-4節 微脂粒………………………………………..37
1-4.1 微脂粒的結構……..………………………………….37
1-4.2 微脂粒的型態……..………………………………….41
1-4.3 微脂粒的穩定性..…………………………………….44
1-4.4 微脂粒的應用……..………………………………….49
第1-5節 穿膜胜肽與細胞溶解性胜肽………………..51
1-5.1 細胞溶解性胜肽與抗菌胜肽..……………………….51
1-5.2 穿膜胜肽(transmembrane peptide)………………..52
1-5.3 穿膜胜肽的膜轉移(membrane translocation)機制……………………………………53
1-5.4 穿膜胜肽(transmembrane peptide)的毒性.………..54
1-5.5 穿膜胜肽與細胞溶解性胜肽的 研究發展………………………………….…………..55
1-5.6 蜂毒胜肽(melittin)…………………..………………..58
1-5.6.1 Melittin的結構……………………...……………..59
1-5.6.2 Melittin的活性……………………...……………..61
1-5.6.3 Melittin於醫學上的應用…………...……………..61


第二章 於金膜表面形成脂雙層膜系統之方法與偵測…………………………………………………..63
第2-1節 前言…………………………………………..63
第2-2節 儀器設備與材料……………………………..63
2-2.1 藥品………………………….………………………..63
2-2.2 儀器設備……………………………………………...64
第2-3節 實驗方法……………………………………..64
2-3.1 微脂粒的製備……………….………………………..64
2-3.2 粒徑量測……………………………………………...65
2-3.3 脂雙層膜的(lipid bilayer)形成……………………….66
2-3.4 AFM影像偵測………………………………………..68
第2-4節 實驗結果……………………………………..70
2-4.1 微脂粒於金膜表面吸附行為之偵測.………………..70
2-4.2 AFM影像偵測……...………………………………...79
2-4.3 結果討論…….………………………………………..84

第三章 蜂毒胜肽Melittin與脂雙層膜間的交互作用機制之探討
第3-1節 前言…………………………………………..86
第3-2節 儀器設備與材料……………………………..86
3-2.1 藥品………………………….………………………..86
3-2.2 儀器設備……………………………………………...86
第3-3節 實驗方法……….……………………………..87
3-3.1 QCM實驗..………………….………………………..87
3-3.2 ITC實驗.....………………….………………………..88
3-3.3 AFM影像偵測……...………………………………...89
3-3.4 粒徑量測....………………….………………………..89
第3-4節 實驗結果……….……………………………..91
3-4.1 QCM實驗..………………….………………………..91
3-4.2 ITC實驗.....………………….………………………105
3-4.3 AFM實驗…………...……………………………….112
3-4.4 DLS實驗....………………….………………………118
3-4.5 結果討論…….………………………………………120

第四章 總結………………………………………...127
參考文獻…………………………………………….129

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