利用原子力顯微鏡測量老鼠普里昂蛋白與乙型類澱粉蛋白 42 共培養之類澱粉樣纖維

Abstract

普里昂蛋白 (PrP) 是引發人類庫賈氏症、狂牛症以及羊搔癢症的主要蛋白質，其異常折疊造成腦組織空洞化。先前的研究指出，普里昂蛋白為乙型類澱粉蛋白 42 (Aβ42) 寡聚物具高親和性的受體。此外，PrP 與 Aβ42 寡聚物的結合被認為與阿茲海默症的惡化過程有密切相關性。為了探討 PrP 與 Aβ42 的相互作用，本研究運用直徑平均為 28 nm 的逆相微胞來共培養 PrP 和 Aβ42 單體，並使用動態光散射儀確認逆相微胞的大小，以及利用硫磺素 T 螢光檢測法和穿透式電子顯微鏡探討在共培養的條件下 PrP 和 Aβ42 能否產生共寡聚物。實驗結果顯示，逆相微胞中的限制性物理空間能增加 PrP 與 Aβ42 寡聚物的相互作用，比單獨培養PrP時形成較多 β-sheet 的結構。本研究進一步使用原子力顯微鏡來量測 PrP、Aβ42 以及共培養 PrP—Aβ42 纖維的高度及硬度值，結果顯示三者的硬度值在統計上並沒有顯著的差異。

Prion protein (PrP) is the culprit that causes human Creutzfeldt-Jakob disease, mad cow disease and scrapie in sheep. Its abnormal folding causes the spongy form of brain tissues in patients. Previous studies have shown that PrP is a high-affinity receptor for the oligomers of beta amyloid 42 peptides (Aβ42). Moreover, the binding between PrP and Aβ42 oligomers is closely associated to the progression of Alzheimer's disease. In order to investigate the interaction between PrP and Aβ42, this study used reverse micelles (RM) with an average diameter of 28 nm to co-incubate PrP and Aβ42 monomers. The size of RM was monitored by dynamic light scattering (DLS), and the interaction between PrP and Aβ42 was investigated by Thioflavin-T (ThT) assay and transmission electron microscopy (TEM). The results showed that the confined physical space of the RM can enhance the binding of PrP and Aβ42 oligomers. The resultant PrP–Aβ42 aggregates had more β-sheet structure than the aggregates formed by PrP alone. We then used atomic force microscopy (AFM) to measure the height and Young’s modulus of the fibrils of PrP, Aβ42 and PrP–Aβ42. The results indicate that the Young's modulus of the three types of fibrils have no significant difference.