以逆相微胞製備乙型類澱粉樣多肽聚合物之研究

乙型類澱粉樣蛋白 (beta amyloid peptides, Aβ) 為阿茲海默症中關鍵因素，近年來研究指出寡聚物 (oligomers) 比纖維狀態具有更高細胞毒性。在本研究中，我們將Aβ培養於2,2,4-三甲基戊烷 (isooctane) 與Aerosol-OT (sodium bis(2-ethylhexyl) sulfosuccinate, AOT) 製備的逆相微胞 (reverse micelle) 中。動態光散射粒徑分析儀 (dynamic light scattering) 顯示其水合直徑約為33 nm。於25 °C培養六天後，反向萃取Aβ胜肽 (RMAβ6d) 以穿透式電子顯微鏡 (transmission electron microscope) 與OMAB抗體辨認，其為寡聚物狀態；以粒徑篩層析儀 (size exclusion chromatography) 分析其最大為54 kDa；Thioflavin-T (ThT) 數據指出具有β-sheet之反向萃取寡聚物快速生長為原纖維。固態核磁共振 (solid-state nuclear magnetic resonance) 結果顯示這些原纖維為單一構型 (monomorphism)。大部分in vitro培養之Aβ纖維具多態性 (polymorphism)，我們推斷主要源自多重成核途徑；在本實驗中，因逆相微胞之物理性限制空間，Aβ聚集由一級成核 (primary nucleation) 主導，因此能培養出結構均一之原纖維聚集物。此發現能解釋不同阿茲海默症患者腦中，有著不同構型之核種，卻由單一聚集生長機制主導，因此產生不同結構之Aβ纖維，但卻保有單一構型。

關鍵字

乙型類澱粉樣胜肽；寡聚物

並列摘要

Beta-amyloid peptides (Aβ) are widely considered as the key factor in the molecular pathology of Alzheimer's disease (AD). According to recent research, Aβ oligomers are considered to be a more relevant therapeutic target than other species such as the fibrillar aggregates. In this study, we were able to incubate Aβ peptides in the reverse micelles (RMs) formed by water, sodium bis(2-ethylhexyl) sulfosuccinate (AOT), and isooctane, where the molar ratio of water to AOT was adjusted to 70. The hydrodynamic diameter of the reverse micelles was found to be ca. 33 nm by dynamic light scattering (DLS). After incubated at 25 °C for 6 days, Aβ peptides were in oligomeric state as confirmed with TEM images and OMAB dot blot assay. The size of the oligomers was estimated with analytical size exclusion chromatography (SEC) to be <54 kDa. The results of Thioflavin-T (ThT) assay revealed that the extracted Aβ oligomers in Tris buffer grew rapidly into protofibrils without lag time. Surprisingly, the solid-state NMR data indicate that the molecular structure of the protofibrils is highly monomorphic. We infer that the phenomenon of structural polymorphism commonly observed in amyloid fibrils prepared in vitro is largely due to the nucleation process of the amyloidogenic peptides, where both the primary and secondary nucleation processes are occurring. In this work, when the nucleation process was dominated by a single molecular process, i.e., primary nucleation within RMs, the molecular structure of the fibrillar aggregates became monomorphic. This finding sheds considerable insight into the intriguing observation that brain tissues of individual AD patients exhibit a single predominant but distinctive Aβ fibrillar structure.