乙型類澱粉樣蛋白（Aβ）的沉積物為阿茲海默症的病理特徵之一，因此被認為是造成阿茲海默症的關鍵原因。為了進行後續藥物與治療方式的開發，我們欲對Aβ蛋白的結構進行研究。然而在過往文獻中，in vitro培養之Aβ蛋白纖維的分子結構呈現多態性（polymorphism）。我們假設纖維的結構多樣性是源於具異質性的Aβ蛋白在依循晶場成核理論聚集時，以多種成核路徑（初成核與各種次成核路徑）聚集所造成的。為了得到Aβ蛋白的單一結構，我們利用逆向微胞作為一個奈米尺度的物理限制空間調控Aβ蛋白的成長，強制使其停留在亞穩態中間體（metastable intermediate）的寡聚物（oligomer）狀態，並以逆向微胞均一分布的特性將寡聚物保持在環境與大小皆相似的狀態下，希望因此能取得結構具高同質性的寡聚物。藉由打破逆向微胞後，以引晶實驗將寡聚物作為核種，將成核方式限制為單一的初成核步驟，以期得到單一構型的Aβ蛋白纖維。而在實驗室前人以Aerosol-OT （AOT）作為逆向微胞製備的Aβ寡聚物中有大量AOT殘留，並會影響Aβ單體的聚合速率，故為了能克服AOT殘留物對後續引晶實驗的干擾，並模擬腦內細胞膜對Aβ蛋白的影響和方便進行細胞毒性的實驗，本研究選擇從大豆中提取的磷脂醯膽鹼作為逆向微胞的材料，成功架設新的逆向微胞系統並進行上述實驗。然而以新制備的寡聚物作為核種引晶時，發現在逆向微胞中培養不同天數的寡聚物樣品具有不同的引晶能力，於生物毒性的實驗上也看到樣品性質差別：在逆向微胞中培養越久的寡聚物樣品引晶能力較弱但生物毒性較強。我們推斷原因可能是在逆向微胞中培養越久的寡聚物會與殘留的磷酯醯膽鹼結合越緊密，造成引晶能力較弱而且細胞毒性較強。

Aggregates of β-amyloid peptide （Aβ） are an important pathological signature of Alzheimer''s disease （AD）. Currently, various Aβ aggregates such as oligomers, protofibrils, and fibrils are widely considered as the key factors controlling the progression of AD. For therapeutic treatments, we need to determine the molecular structure of Aβ aggregates. However, Aβ aggregates prepared in vitro are highly polymorphic. We hypothesize that the structure polymorphism is a direct consequence of the coexistence of various nucleation pathways, viz., the primary nucleation, fibril fragmentation, and the fibril-assisted secondary nucleation. In order to get a monomorphic structure of Aβ fibrils, we attempt to use nano-sized reverse micelle as a physically confined space to incubate Aβ peptides so that the peptides are trapped into their oligomeric state. After the backward extraction of the peptides from the reverse micelles, oligomers could be used to seed the fibrillization of Aβ monomers. In this way, Aβ monomers could aggregate mainly through the primary nucleation pathway, from which we should be able to get monomorphically structured Aβ fibrils. In our earlier studies, the surfactant, Aerosol-OT （AOT） was used to prepare reverse micelles to encapsulate Aβ peptides. Unfortunately, the residual AOT after backward extraction would interfere the aggregation kinetics of Aβ monomers. In this study, we therefore chose the phosphatidylcholine extracted from soybean, which is a bio-compatible amphiphilic molecule, to prepare our reverse micelles. We aim to verify our hypothesis on the structural polymorphism of Aβ aggregates by this newly developed reverse micelles. In the subsequent seeding experiments, the oligomers of different incubation time in PC-RM show different properities. Compared to the oligomers of shorter incubation time, oligomers with longer incubation time in PC-RM tend to have a minor seeding ability but stronger toxicity in cell vability test. We suggest that the results are cause by stronger interactions between the lipid molecules and the Aβ oligomers for longer incubation time in PC-RM.

口試委員審定書 II
中文摘要 i
Abstract ii
縮寫表 iv
目錄 vi
圖目錄 ix
表目錄 xiv
1. 第一章 緒論 1
1.1. 阿茲海默症 1
1.2. 類澱粉樣蛋白纖維 2
1.3. 阿茲海默症與類澱粉樣蛋白關聯性 3
1.4. 類澱粉樣蛋白成核路徑 6
1.5. 類澱粉樣蛋白纖維分子結構多樣性 7
1.6. 類澱粉樣蛋白寡聚物 12
1.6.1. 寡聚物培養與種類 12
1.7. 逆向微胞簡介 17
1.8. 研究動機 22
2. 第二章 合成與鑑定 23
2.1. 化學試劑與使用儀器 23
2.1.1. 化學試劑 23
2.1.2. 使用儀器 25
2.2. 胜肽製備 26
2.2.1. 蛋白表達 26
2.2.2. 蛋白純化 29
2.2.3. 蛋白鑑定 31
2.3. 逆向微胞製備 35
2.3.1. Aβ40單體製備 35
2.3.2. 包覆Aβ40逆向微胞製備 35
2.3.3. Aβ40反相萃取 37
2.4. 鑑定方法 38
2.4.1. 動態光散射粒徑分析儀 38
2.4.2. 斑點印跡法 39
2.4.3. 硫磺素螢光偵測 40
2.4.4. 圓偏光二色光譜 41
2.4.5. 穿透式電子顯微鏡 43
3. 第三章 實驗結果與討論 45
3.1. 胜肽之鑑定 45
3.2. 包覆Aβ40之磷脂逆向微胞系統 47
3.2.1. 磷脂逆向微胞系統穩定度 47
3.2.2. 磷脂逆向微胞內Aβ40聚合情形 47
3.3. PC-RM Aβ40寡聚物鑑定 49
3.3.1. 反向萃取之寡聚物TEM鑑定 49
3.4. Aβ40寡聚物引晶實驗控制組 52
3.4.1. Aβ40單體自聚集確認 52
3.4.2. 反向萃取效率確認 53
3.4.3. 磷脂質與界面活性劑控制組 55
3.4.4. PC-RMAβ40寡聚物控制組 56
3.5. Aβ40寡聚物引晶實驗 58
3.5.1. PC-RMAβ40寡聚物對Aβ40單體1:10引晶實驗 58
3.5.2. PC-RMAβ40寡聚物對Aβ40單體1:1引晶實驗 61
3.6. PC-RMAβ40-xd寡聚物細胞實驗 63
3.7. PC-RMAβ40-xd寡聚物結果討論 65
4. 第四章 結論與未來展望 66
4.1. 論文總結 66
4.2. 未來展望 67
5. 參考文獻 69
6. 附錄 77
附錄A 77
附錄B 93
附錄C 108
附錄D 110
附錄E 111
附錄F 118
附錄G 120
附錄H 128

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