臺灣博碩士論文加值系統

阿茲海默症(Alzheimer’s disease; AD)是一種神經退化性疾病。患者主要病理特徵為神經細胞外有類澱粉斑塊的沉積以及神經細胞內之纖維糾結。類澱粉斑塊的主要成份為beta-類澱粉胜肽(Ab。聚集的A�狳蒟姜g毒性，目前認為這是造成阿茲海默症之主因。若能得知Ab聚集過程之分子機制，將有助於我們設計聚集作用的抑制劑，減緩因Ab聚集所產生的傷害。為了解Ab聚集之分子機制，本實驗室先前已利用循理式突變，設計出Ab40之突變種Ab40(L17A/F19A)，並利用核磁共振光譜法探討其結構及聚集之特性。在SDS環境下Ab40(L17A/F19A)不會發生去摺疊(unfolding)及聚集作用，其Q15~D23區域的��-helical propensity 明顯地較原生種Ab40提升許多。在水溶液中，Ab40(L17A/F19A)聚集的速率亦遠低於原生種Ab40，且其細胞毒性亦較原生種Ab40低。從這些特性我們推測Ab40(L17A/F19A)在脂質環境下可能亦較原生種Ab40不易發生去摺疊及聚集。本研究主要目的是利用Ab40(L17A/F19A)較不易聚集的特性，從結構的觀點探討其在脂質環境下聚集之分子機制，藉此幫助我們對原生種Ab40在脂質環境下之聚集機制有更深一層之了解。

Alzheimer’s disease (AD) is a neurodegenerative disease which leads to progressive dementia and neuronal death. The main histopathological hallmarks of AD are the senile plaques within the cerebral cortex and the neurofibrillary tangles within the nerve cells. The primary component of senile plaques is beta-amyloid peptide (Ab).�nRecent studies have suggested that Ab has neurotoxic properties in an aggregated state. Thus, knowing the molecular mechanism of the aggregation process of Ab may facilitate the design and development of fibrillogenesis inhibitors. Previously, we have characterized structural and aggregation properties of a rationally mutated Ab40, Ab40(F17A/L19A), by using NMR spectroscopy in our lab. In SDS environment, the aggregation rate of Ab40(F17A/L19A) is much slower than that of wild-type Ab40. The alpha-helical propensity of Ab40(F17A/L19A) is higher than that of wild-type Ab40 in the region from Q15 to D23. In aqueous solution, the cell toxicity and aggregation rate of Ab40(F17A/L19A) are also lower than those of wild-type Ab40. These characteristics suggest that Ab40(L17A/F19A) may aggregate at a lower rate in a lipid environment. In this study, we will take advantage of these characteristics and apply NMR spectroscopy to investigate the aggregation mechanism of Ab40(L17A/F19A) in lipid environment from the structural point of view. The results may help us gain more insight to the aggregation mechanism of wild-type Ab40.

目錄

目錄 2
圖表目錄 4
附錄目錄 5
中文摘要 6
英文摘要 7
第一章 緒論 8
第二章 材料與方法 12
一、藥品試劑 12
二、核磁共振實驗 12
2-1�n���{類澱粉胜肽之製備 12
2-2核磁共振樣品之製備 13
(a) 脂質樣品製備 13
(b) 水溶液樣品製備 13
2-3投影重建核磁共振實驗 14
2-4蛋白質骨架原子化學位移量測 14
2-5 ���{類澱粉胜肽二級結構之判定 15
第三章 結果與分析 17
一、原生種A��40�n在脂質環境下之2D 1H-15N HSQC光譜圖 17
二、比較A��40�n(L17A/F19A)與原生種A��40在脂質環境下之2D 1H-15N HSQC光譜 17

三、比較A��40(L17A/F19A)在脂質環境與水溶液環境下之2D 1H-15N HSQC光譜圖 18
四、A��40(L17A/F19A)在脂質環境下之共振峰判定與二級結構判定 18
五、比較A��40(L17A/F19A)在脂質環境下不同時間點之2D 1H-15N HSQC光譜圖 19
第四章 討論 21
參考文獻 23





















圖表目錄
圖一、原生種A��40在脂質環境下之2D 1H-15N HSQC光譜圖 25
圖二、原生種A��40�n與A��40�vL�������}F�������w在脂質環境下之2D
1H-15N HSQC光譜重疊圖 26
圖三、����40�vL�������}F�������w在脂質環境與水溶液環境下之2D
1H-15N HSQC光譜重疊圖 27
圖四、A��40(L17A/F19A)在脂質環境下之2D 1H-15N HSQC光譜圖 28
圖五、A��40(L17A/F19A)在脂質環境下不同時間點之光譜比較圖 29
圖六、A��40(L17A/F19A)從E3到F20之3D HNCA條狀圖譜 30
圖七、A��40(L17A/F19A)從A21到M35之3D HNCA條狀圖譜 31
圖八、A��40(L17A/F19A)從V36到V40之3D HNCA條狀圖譜 32
圖九、A��40(L17A/F19A)之13C�悀ずレ麮凝t之長條圖 33
圖十、A��40(L17A/F19A)之13C�狺ずレ麮凝t之長條圖 34
圖十一、A��40(L17A/F19A)之13C’化學位移差之長條圖 35
圖十二、A��40(L17A/F19A)之H�悀ずレ麮凝t之長條圖 36
圖十三、����40(L17A/F19A)�w之化學位移指數 37
圖十四、A��40(L17A/F19A)變化前與變化後之13C�悀ずレ麮凝t之
比較圖 38
表一、����40(L17A/F19A)�w溶於50 mM Tris buffer (336 mM DHPC/DMPC, q = 2, pH 7.0 )之核磁共振實驗參數 39
表二、原生種����40溶於50 mM Tris buffer (336 mM DHPC/DMPC, q = 2, pH 7.0 )之核磁共振實驗參數 39
表三、����40(L17A/F19A)溶於50 mM Tris buffer (336 mM
DHPC/DMPC, q = 2, pH 7.0 )之1H、15N、和13C化學位移 40




附錄目錄
附錄一、A��40(L17A/F19A)從A2到A19之3D CBCA(CO)NH條
狀圖譜 41
附錄二、A��40(L17A/F19A)從F20到L34之3D CBCA(CO)NH條狀圖譜 42
附錄三、A��40(L17A/F19A)從M35到V39之3D CBCA(CO)NH條狀圖譜 43
附錄四、A��40(L17A/F19A)從A2到A19之3D HNCO條狀圖譜 44
附錄五、A��40(L17A/F19A)從F20到L34之3D HNCO條狀圖譜 45
附錄六、A��40(L17A/F19A)從M35到V39之3D HNCO條狀圖譜 46
附錄七、A��40(L17A/F19A)從A2到A19之3D HBHA(CO)NH條狀圖譜 47
附錄八、A��40(L17A/F19A)從F20到L34之3D HBHA(CO)NH 48
附錄九、A��40(L17A/F19A)從M35到V39之3D HBHA(CO)NH 49
附錄十、二十種胺基酸之13C�恁B13C�牷B13C’和�？�悀G級結構傾向化學位移參考值 50

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