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標題: | 以磷脂逆向微胞製備乙型類澱粉樣蛋白寡聚物及其衍生之纖維 Preparation of beta-amyloid oligomers and their derived fibrils in phospholipid reverse micelles |
作者: | Tzu-Yun Lin 林子筠 |
指導教授: | 陳振中(Jerry Chun Chung Chan) |
關鍵字: | 阿茲海默症,乙型類澱粉樣蛋白,寡聚物,大豆磷脂醯膽鹼,逆向微胞,引晶實驗,細胞毒性,蛋白纖維, Beta-amyloid peptide,oligomer,phosphatidylcholine,reverse micelle,seeding experiment,cell toxicity,Aβ fibrils, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/73944 |
DOI: | 10.6342/NTU201903441 |
全文授權: | 有償授權 |
顯示於系所單位: | 化學系 |
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