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標題: | 以逆相微胞控制乙型類澱粉蛋白寡聚物大小並探討其細胞毒性及結構差異 Size control of Aβ40 oligomers by reverse micelles and study of their difference in cytotoxicity and structure |
作者: | 葉貞岑 Chen-Tsen Yeh |
指導教授: | 陳振中 Jerry Chun-Chung Chan |
關鍵字: | 乙型類澱粉蛋白寡聚物,逆相微胞,細胞毒性,動態變化,固態核磁共振, Aβ oligomers,reverse micelle,cytotoxicity,motional dynamics,solid-state NMR, |
出版年 : | 2023 |
學位: | 碩士 |
摘要: | 阿茲海默症是一種神經退化性疾病,早期研究認為在大腦中堆積的乙型類澱粉蛋白 (Aβ) 斑塊是造成阿茲海默症的成因,近年來則有研究顯示出Aβ寡聚物具有神經細胞毒性,並且對於神經突觸造成傷害。最新設計的單克隆抗體Lecanemab,結合可溶性Aβ聚集體的能力遠大於單體及纖維,相較於過去偏好結合纖維或單體的抗體,Lecanemab能夠更有效減少患者腦中的斑塊沉積以及減緩認知退化,顯示出Aβ寡聚物為靶向治療關鍵,因此Aβ寡聚物為此論文的研究重點。但因為Aβ會快速聚集形成纖維的特性,在過去的文獻中很難對Aβ寡聚物進行系統性研究,至今仍沒有研究指出寡聚物的毒性與結構之間的關係,也無法了解實際的致病機制。
過去本實驗室以cyclohexane/CO520/NH4OAc三相系統組成的逆相微胞製備出23 nm Aβ40寡聚物,命名為RM23-Aβ40,本研究將油相改為使用1:1混合的n-hexane及cyclohexane,再進一步調整界面活性劑濃度以及水跟界面活性劑的比例,製備出10 nm的逆相微胞,將Aβ40單體限制於10 nm逆相微胞中,透過微胞間的碰撞使得微胞內單體聚集形成Aβ40寡聚物。利用穿透式電子顯微鏡拍攝從10 nm逆相微胞中反萃出的Aβ40寡聚物,命名為RM10-Aβ40,後續利用ImageJ批次定量分析Aβ40寡聚物平均直徑為8.7 ± 2.8 nm,再使用硫磺素 (ThT) 螢光分析,顯示出RM10-Aβ40會自聚集形成纖維且有一段RM23-Aβ40沒有的延滯期,代表RM10-Aβ40需經過結構的轉變才能形成纖維,顯示出RM10-Aβ40與RM23-Aβ40為纖維化過程中不同階段的蛋白質寡聚物,且RM10-Aβ40為較小較早期的Aβ40寡聚物。再藉由alamarBlue assay比較兩種寡聚物的細胞毒性,結果顯示RM10-Aβ40相較於RM23-Aβ40具有更高的毒性,可能是造成阿茲海默症的關鍵蛋白。為了瞭解RM10-Aβ40的結構資訊,我們使用固態核磁共振進行測量。結果顯示RM23-Aβ40和RM10-Aβ40整體的化學位移及半峰全寬大約相同,但從訊號強度分析顯示出RM10-Aβ40的帶電荷殘基動態變化程度高於RM23-Aβ40,推測殘基高度動態變化可能是影響細胞毒性及纖維化過程的關鍵。本研究比較了不同大小Aβ40寡聚物毒性與結構差異,推測Aβ40寡聚物的殘基動態變化程度與細胞毒性可能更具相關性。 Alzheimer's disease (AD) is a neurodegenerative disease. Early research suggested that the accumulation of beta-amyloid (Aβ) protein plaques in the brain was the cause of AD. However, recent studies have shown that Aβ oligomers have neurotoxicity and impair synaptic function. The latest monoclonal antibody, Lecanemab, designed to bind the soluble Aβ aggregates, is more effective in reducing brain Aβ plaques and slowing cognitive decline compared to previous antibodies, which preferentially bind to fibrils or monomers. This demonstrates that targeting Aβ oligomers is crucial to curing AD. Therefore, Aβ oligomers are the current focus of research. However, due to the characteristic of Aβ rapidly aggregating to form fibers, it has been difficult to study Aβ oligomers in the past. To date, no studies have identified the relationship between the toxicity and structure of Aβ oligomers. In the past, we prepared 23 nm Aβ40 oligomers using a reverse micelle (RM) system composed of a cyclohexane/CO520/NH4OAc ternary system, which we called RM23-Aβ40. In this study, we changed the organic phase to a 1:1 mixture of n-hexane and cyclohexane and further adjusted the surfactant concentration and the water to surfactant ratio for the preparation of 10-nm RMs. The Aβ40 monomers were confined to the RMs and aggregated to form Aβ40 oligomers through collisions between micelles. Transmission electron microscopy (TEM) images showed the morphology of the Aβ40 oligomers extracted from the 10-nm RMs, which we named RM10-Aβ40. Then we used ImageJ to quantitative the size was 8.7 ± 2.8 nm. Subsequently, thioflavin T (ThT) fluorescence analysis showed that RM10-Aβ40 can self-aggregate to form fibers and had a lag phase that RM23-Aβ40 did not have. This indicates that RM10-Aβ40 needs structural transformation to form fibers, while RM23-Aβ40 can form fibers without structural transformation. It showed that RM10-Aβ40 and RM23-Aβ40 are Aβ40 oligomers at different stages of the fibrillization process, and RM10-Aβ40 is at the early stage. From the results of alamarBlue assay, we observed that the cytotoxicity of RM10-Aβ40 was higher than RM23-Aβ40, indicating that it may be a crucial species contributing to AD. To further investigate their structural information, we carried out solid-state NMR measurements on 13C labeled RM10-Aβ40. Even though the overall line widths and chemical shifts of the RM10-Aβ40 and RM23-Aβ40 were comparable, intensity analysis revealed a higher degree of motional dynamic in charged residues of RM10-Aβ40 than in RM23-Aβ40. This study directly compared the cytotoxicity and structural differences between different size of Aβ40 oligomers. Overall, we suggest that the motional dynamics might have stronger correlation with cytotoxicity. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/88585 |
DOI: | 10.6342/NTU202302296 |
全文授權: | 同意授權(限校園內公開) |
顯示於系所單位: | 化學系 |
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