利用表面電漿共振及單分子模型槽探討類澱粉胜?與不同組成微脂粒交互作用動力學之研究; Kinetics Studies of Interactions between β-amyloid and Various Composition Liposome by Surface Plasmon Resonance and Langmuir-Blodgett Trough

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题名:	利用表面電漿共振及單分子模型槽探討類澱粉胜?與不同組成微脂粒交互作用動力學之研究;Kinetics Studies of Interactions between β-amyloid and Various Composition Liposome by Surface Plasmon Resonance and Langmuir-Blodgett Trough
作者:	范芙蓉;Fu-Jung Fan
贡献者:	化學工程與材料工程研究所
日期:	2006-06-20
上传时间:	2009-09-21 12:26:10 (UTC+8)
出版者:	國立中央大學圖書館
摘要:	本研究主要是探討 Aβ? 與微脂粒之交互作用，以不同培養時間的 Aβ 與不同組成之微脂粒（使用DPPC、DPPG 及添加神經節苷酯（GM1）及膽固醇等）進行討論，以期能說明 Aβ? 與細胞膜之作用行為。實驗上主要分為兩部分：第一部份利用單分子模型槽探討 Aβ? 與單分子層之間隨時間表面壓的變化；第二部分是利用表面電漿共振儀及恆溫滴定微卡計探討 Aβ? 與微脂粒交互作用之動力學及熱力學。另，也配合原子力顯微鏡來偵測SPR 實驗後其Aβ 及微脂粒的構型。使用Aβ (1-40)溶液與不同組成之單分子層的交互作用，本研究先建立不同單分子膜組成之Π-A 圖，且選定初始表面壓為 15mN/m，來量測Aβ 分子由溶液擴散至氣/液界面的速率。其速率：帶有負電的脂質（DPPG 或GM1）>DPPC+膽固醇>兩性脂質（DPPC），顯示一開始吸附於單分子層是靜電作用力為主。而吸附至氣/液界面的 Aβ 分子結構的重排與構型轉變行為速率為：同時帶有負電脂質（DPPG）及神經節苷酯（GM1）+膽固醇>帶負電脂質>DPPC+膽固醇>DPPC，表示同時有負電脂質跟膽固醇存在時，有助於Aβ 於膜上的構型轉變。在表面電漿共振儀探討 Aβ (1-40)與微脂粒交互作用之動力學研究方面，單體的 Aβ 與不同組成微脂粒作用其親和力大小為： DPPC/GM1/膽固醇（5:3:2）>DPPC/膽固醇（7:3）>DPPC/DPPG/膽固醇（5:2:3）>DPPC/膽固醇（9:1）>DPPC。由親和力強度可判斷單體的 Aβ 與不同組成之微脂粒交互作用主要是靠靜電作用力。量測聚集體與微脂粒交互作用，發現聚集體的Aβ 其親和力較單體的Aβ 低，表示聚集體的Aβ 較不易與微脂粒作用。而在單體 Aβ? (1-40)與微脂粒交互作用之熱力學研究方面，利用 ITC 量測單體Aβ 與DPPC、DPPC+膽固醇及DPPC/DPPG/膽固醇(5:2:3) 之交互作用時，主要為放熱反應，結果顯示其作用力主要為靜電吸引力。若添加膽固醇之微脂粒與Aβ作用，使Aβ 較易形成β 結構及膜與 Aβ 吸附區域形成之cluster 更易誘發溶液中Aβ 與膜表面的Aβ聚集，且因疏水作用力反應漸漸變成吸熱。若同時添加膽固醇與負電脂質之微脂粒系統與Aβ作用，其P/L 大於0.0023 時，膜表面的Aβ會慢慢誘發溶液中的Aβ 與其聚集而放熱變小，而DPPC 微脂粒系統，其P/L 需到達0.0045 ，才會開始吸引溶液中的Aβ 與其聚集。表示帶有負電脂質與膽固醇同時存在時，有助於膜上的Aβ 誘發溶液中的Aβ 於膜表面聚集。 β-amyloid（Aβ）was believed to cause the prime factor of Alzheimer’s disease. However, the mechanism of the cytotoxicity and the disease caused by Aβ is still unclear. The objective of this work is to study the interactions of various incubation time of Aβ with designed liposomes. The objectives were achieved by the following studies : First at all, the adsorption kinetics behaviors of Aβ on lipid monolayers were studied by NIMA trough. Secondly, surface plasmon resonance (SPR) and isothermal titration microcalorimetry (ITC) to measure the kinetics and binding enthalpy of the interactions between Aβ and various composition liposomes. The morphology of liposome before and after interaction with Aβ were monitored by atomic force microscopy (AFM). Results from lipid monolayer trough studies showed that the rate of Aβ adsorbed onto lipid monolayer is mainly due to the electrostatic effect, and the structural rearrangements of the adsorbed Aβ is sensitive to the lipid monolayer composition. Kinetics of Aβ adsorption onto the liposomes by SPR reveal also that the driving force of fresh Aβ interacts with various liposomes is electrostatic interactions. Fresh and monomer type of Aβ is shown to have higher affinities for GM1 containing liposome than that of aggregated Aβ. Addition of cholesterol to the liposome could alter membrane fluidity and promote the fresh Aβ in solution to interact with Aβ onto the membrane. The binding enthalpies between fresh Aβ and liposomes measured by ITC are exothermic reactions and are contributed to the electrostatic interaction. The effects of peptide/lipid molar ratio(P/L) on the binding enthalpy were also discuss. In general, the binding enthalpy become less exothermic as P/L ratio increase, and the transition happens in lower P/L ratio for charged lipid.
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