臺灣博碩士論文加值系統

由β-amyloid (Aβ)胜&;#32957;所組成的類澱粉樣纖維沉積物為阿茲海默症最明顯的病理特徵之一。Aβ從單體聚合成類澱粉樣纖維的過程之中，寡聚物被視為是重要的中間態，並且被認為很可能是造成細胞死亡的元凶之一。然而，由於寡聚物的定義不明確並且合成純化困難，實驗上並不容易得到均勻度高的樣品以進行分子結構的測量。因此，我們嘗試以微脂體提供物理性的空間，將Aβ1&;#8722;40限制在其中生長，期望能阻止其形成纖維，並在微脂體內達到穩定一致的結構，以便使用固態核磁共振技術分析結構。
在本實驗當中，我們使用逆相蒸發法合成微脂體來包覆Aβ1&;#8722;40胜&;#32957;單體，其包覆效率為14%左右，比一般常用的薄膜水合法來得高。在經過培養之後，我們在電子顯微鏡下觀察到類澱粉纖維的形貌，顯示微脂體並無法限制住類澱粉纖維的生長，反而是促進Aβ1&;#8722;40在低濃度(3 μM)的條件下生長。我們認為其原因可能是限制空間保持了較高的Aβ1&;#8722;40局部濃度，同時微脂體的膜表面可做為二維的模板以促進Aβ1&;#8722;40的纖維化。微脂體培養出的纖維以及在低溫、溶液態中培養出來的Aβ1&;#8722;40寡聚物經由固態核磁共振技術分析之後，均具有β-sheet的二級結構，但在分子結構上有些許差異，與文獻中Tycko所發表的Aβ1&;#8722;40纖維的結構也不同。此結構差異可能來自於微脂體對纖維結構的調控，但仍需要進一步證實。

One of the hallmarks of the Alzheimer’s disease (AD) is the amyloid plaques consisting of β-amyloid (Aβ) fibrils. Aβ oligomers have been considered as an important intermediate in the pathway of fibrillization, and are suggested to be the primary pathological species of AD. However, little is known about the molecular structure of Aβ oligomers because of their structural heterogeneity and transient nature. Therefore, we attempt to use liposomes to limit the fibrillization process by providing a confined space for Aβ1&;#8722;40 in order to obtain stabilized oligomers, for which solid-state NMR technique can be utilized to identify the molecular structure.
In this work, liposomes containing approximately 14% of Aβ1&;#8722;40 monomers are synthesized with the reverse-phase evaporation method, which is more efficient than the thin-film liposome preparation method. However, instead of being confined by liposomes, the peptides form fibrils at a surprisingly low concentration of 3 μM. We propose that the liposomes may maintain a high local concentration of peptides, and may act as two-dimensional templates to accelerate the fibrillization of Aβ1&;#8722;40. Solid-state NMR spectra reveal that the fibrils possess β-sheet secondary structure which is structurally different from oligomers prepared in buffer solution at 4 degrees Celsius. The chemical shift data of our fibril sample show significant difference from those reported for Tycko’s fibrils incubated in bulk solution. Additional experiments are required to confirm whether or not the structural distinction of our fibril sample is due to the modulation effect of liposomes.

謝誌……. i
中文摘要 iii
Abstract… iv
縮寫表… v
第一章 緒論 1
1-1 類澱粉樣蛋白纖維 1
1-2 阿茲海默症以及Aβ胜&;#32957;所扮演的角色 5
1-3 不同形態的Aβ寡聚物 8
1-4 以固態核磁共振光譜探討Aβ聚合體之結構 10
1-5 Aβ寡聚物之穩定 14
1-6 微脂體及其製備方法之簡介 15
1-7 Aβ胜&;#32957;與脂質膜間的交互作用 18
1-8 研究動機 20
1-9 參考資料 20
第二章 合成與鑑定 29
2-1 材料與使用儀器 29
2-1-1 化學藥品 29
2-1-2 實驗儀器 31
2-2 胜&;#32957;製備 33
2-2-1 胜&;#32957;合成 33
2-2-2 胜&;#32957;純化 36
2-2-3 胜&;#32957;鑑定 37
2-3 Aβ1-40類澱粉樣纖維及寡聚物製備 39
2-4 包覆Aβ1-40單體之微脂體製備 40
2-4-1 LipoAβ製備—薄膜水合法 40
2-4-2 LipoAβ製備—逆相蒸發法 42
2-4-3 LipoAβ純化—凝膠管柱層析法 43
2-5 Aβ1-40類澱粉樣纖維、寡聚物及微脂體之鑑定 43
2-5-1 ThT螢光偵測 43
2-5-2 穿透式電子顯微鏡 45
2-5-3 動態光散射粒徑分佈儀 46
2-6 微脂體包覆率之檢測—斑點印&;#36857;法 48
2-7 Aβ1-40於微脂體內部之結構鑑定—固態核磁共振光譜 49
2-7-1 核磁共振基本原理 50
2-7-2 固態核磁共振技術 51
2-8 參考文獻 56
第三章 實驗結果與討論 59
3-1 胜&;#32957;的合成、純化與鑑定 59
3-2 Aβ1-40類澱粉樣纖維及寡聚物之鑑定 61
3-3 以微脂體包覆Aβ1-40單體 65
3-3-1 lipoAβ中Aβ1-40之含量 67
3-3-2 LipoAβ樣品中微脂體之鑑定 70
3-3-3 LipoAβ樣品中Aβ1&;#8722;40之鑑定 73
3-4 Aβ1&;#8722;40分子結構之鑑定&;#8722;固態核磁共振光譜 76
3-5 結果討論 84
3-6 參考資料 86
第四章 結論及未來展望 89
4-1 論文總結 89
4-2 未來展望 89
4-3 參考資料 90
附錄……. 92
(一) 同位素標定之Aβ1&;#8722;40鑑定 92
(二) 微脂體之Aβ1&;#8722;40局部濃度計算 96

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