王勝仕臺灣大學：化學工程學研究所劉冠男Liu, Kuan-NanKuan-NanLiu2007-11-262018-06-282007-11-262018-06-282005http://ntur.lib.ntu.edu.tw//handle/246246/52236目前發現，人體內至少有20種不同的蛋白質，與致病性的澱粉沈澱(amyloid deposits)的纖維組成(fibrillar components)有關。而因蛋白質發生大小或形狀上的變化，產生聚集，進一步造成類澱粉症(amyloidosis)，也就是蛋白質構象病(protein conformational disease)；其中，類澱粉β蛋白質(β-amyloid, Aβ)為導致阿滋海默症(Alzheimer's Disease)之主因，亦為這些蛋白質中相當重要之一種。 文獻顯示，類澱粉β蛋白質-細胞膜間交互作用與類澱粉β蛋白質誘發之神經毒性高度相關。研究亦發現，此交互作用可能受靜電作用力或細胞膜流動性所影響。然而，確定主宰此類澱粉β蛋白質與細胞膜間交互作用之機制至今仍無定論。 本研究提出：類澱粉β蛋白質-細胞膜間交互作用受與細胞膜性質息息相關之偶極電位(dipole potential)所影響之假說。認為偶極電位之變化反映類澱粉β蛋白質與細胞膜間之結合(binding)情形，而更進一步影響到誘發之毒性機制。因此吾人利用電場敏感性染劑(potential-sensitive fluorescent dye)配合雙波長螢光比值檢測法(dual-wavelength ratiometric fluorescence method)，量測細胞膜偶極電位受不同實驗條件變化之影響；並進一步觀察細胞膜偶極電位之改變，以對其間之交互作用作深入之探討與了解。結果顯示，當聚集狀態下之類澱粉症蛋白質與微脂粒作用時，可使其偶極電位下降；除此之外，添加膽固醇對於類澱粉β蛋白質所引起偶極電位下降之現象更加明顯。而吾人相信，本研究之結果可提供對於阿滋海默症及其他類澱粉症之機制更多的了解Up to now, at least twenty different human proteins and peptides have been isolated as the fibrillar components of disease-associated amyloid deposits. These proteins undergo aberrant changes in structure and accumulate to form aggregated species which lead to the amyloidogenic diseases. β-amyloid peptide (Aβ), the principal protein of Alzheimer’s disease, is also one of them. Previous studies showed that the interaction between Aβ and cell membrane is highly correlated to Aβ-elicited neurotoxicity. Several lines of evidence showed that the interaction between phospholipid membrane and Aβ might be associated with electrostatic-mediated forces or membrane fluidity. However, the detailed mechanism of this interaction remains largely unknown. In this work, we proposed a hypothesis that there is a link between membrane dipole potential and Aβ-cell membrane interaction, which is considered to be the first step in the mechanism of Aβ-induced toxicity and wish to test this hypothesis. To pursue our goals, we employed a dual-wavelength ratiometric fluorescence method with aid of the potential-sensitive fluorescent dye. At the outset, we sought out the optimal experimental conditions of the aforementioned ratiometric method. Next, we investigated the effect of binding of amyloid proteins and phospholipid vesicle on membrane dipole potential. Our results indicated that a decrease in dipole potential was observed upon binding of aggregated amyloid proteins to phospholipid membrane. In addition, a more dramatic change in membrane dipole potential was detected in phospholipid vesicles with cholesterol. We believe that our outcome may contribute to a better understanding of the mechanism(s) associated with AD and other amyloid diseases.第一章 緒論 1 第二章 文獻回顧 3 2-1 阿滋海默症(Alzheimer’s disease, AD)介紹 3 2-1-1 類澱粉症(Amyloidosis) 3 2-1-2 失智(dementia)與阿滋海默症(Alzheimer’s disease, AD) 4 2-1-3 阿滋海默症的分類 7 2-1-4 阿滋海默症的主要成因假說 8 2-2 阿滋海默症與類澱粉β蛋白質(β-amyloid, Aβ) 10 2-2-1 類澱粉前驅蛋白(amyloid precursor protein) 10 2-2-2 類澱粉β蛋白質 14 2-3 類澱粉β蛋白質與細胞膜間之交互作用 26 2-3-1 自由基生成(free radical generation) 26 2-3-2 鈣離子濃度平衡(calcium homeostasis) 28 2-3-3 類澱粉β蛋白質對細胞膜流動性(membrane fluidity)的影響 30 2-3-4 離子通道生成(ion channels formation) 31 2-3-5 靜電作用力對類澱粉β蛋白質與細胞膜交互作用之影響 32 2-3-6 類澱粉β蛋白質與細胞膜交互作用總結 33 2-4 微脂粒(Liposome)介紹 33 2-4-1 微脂粒簡介 33 2-4-2 微脂粒的分類 35 2-4-3 微脂粒的製備方法 35 2-4-4 微脂粒的安定性 39 2-5 偶極電位(Dipole potential)介紹 42 2-5-1 細胞膜(cell membrane)簡介 42 2-5-2 膜電位(membrane potential)介紹 44 2-5-3 膜電位之測量方法 49 第三章 研究動機 52 第四章 實驗裝置、藥品與步驟 53 4-1 實驗裝置 53 4-2 藥品 54 4-3 實驗步驟 55 4-3-1 PBS (phosphate buffered saline)之製備 55 4-3-2 微脂粒之製備 56 4-3-3 類澱粉β蛋白質Aβ(25-35)之備製 56 4-3-4 類澱粉β蛋白質Aβ(1-40)之備製 57 4-3-5 確實形成聚集狀態之類澱粉β蛋白質Aβ(1-40)備製 57 4-3-6 溶菌酶(lysozyme)之備製 58 4-3-7 雙波長螢光比值檢測法(dual-wavelength ratiometric fluorescence method) 58 第五章 結果討論 65 5-1 雙波長螢光比值檢測法(dual-wavelength ratiometric fluorescence method) 66 5-2 靜置時間之影響 70 5-3 不同溶劑對R值的影響 80 5-4 不同微脂粒濃度對R值的影響 83 5-5 類澱粉β蛋白質片段Aβ(25-35) 對細胞膜偶極電位的影響 85 5-6 類澱粉β蛋白質片段Aβ(1-40)對細胞膜偶極電位的影響 89 5-7 溶菌酶(hen agg white lysozyme)對細胞膜偶極電位的影響 93 5-8 膽固醇(cholesterol)與各物種和細胞膜偶極電位的關係 96 5-9 聚集狀態(aggregation state)之測試 100 5-10 總結：各物種和細胞膜偶極電位的關係 112 第六章 結論與建議 119 第七章 參考文獻 1211107065 bytesapplication/pdfen-US類澱粉症偶極電位阿滋海默症amyloidosisdipole potential[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/52236/1/ntu-94-R92524082-1.pdf