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標題: | 以微脂體限制類澱粉樣多肽分子之固態核磁共振光譜結構探討 Confinement of Aβ1−40 Peptides in Liposome for Structural Characterization by Solid-State NMR |
作者: | Chien-I Yang 楊千儀 |
指導教授: | 陳振中(Jerry Chun Chung Chan) |
關鍵字: | 阿茲海默症,類澱粉樣蛋白,固態核磁共振,微脂體, beta-amyloid,solid-state NMR,liposome, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 由β-amyloid (Aβ)胜肽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Aβ1−40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Aβ1−40胜肽單體,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Aβ1−40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Aβ1−40局部濃度,同時微脂體的膜表面可做為二維的模板以促進Aβ1−40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Aβ1−40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Aβ1−40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 Aβ1−40 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 Aβ1−40 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 Aβ1−40. 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. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/56809 |
全文授權: | 有償授權 |
顯示於系所單位: | 化學系 |
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