臺灣博碩士論文加值系統

阿茲海默症為一種最常見的慢性神經退化疾病，主要影響著老年族群。先前研究發現β-類澱粉胜肽為類澱粉斑塊(amyloid plaque)的主要成分，而類澱粉斑塊是現在病理解剖確認為阿茲海默症的一個重要指標。β-類澱粉蛋白(Aβ)是由39-42個胺基酸所組成的短肽鏈，由類澱粉前驅蛋白水解代謝後產生。造成阿茲海默症的假說有很多種，而現今最被廣為接受的的假說為"類澱粉蛋白鏈鎖聚合假說(Amyloid cascade hypothesis)。此假說認為Aβ聚集堆積使腦神經細胞死亡為造成阿茲海默症主要的原因。有研究發現
β-類澱粉胜肽的聚集堆積與細胞膜的交互作用有關，然而β-類澱粉胜肽聚集之機制到現在並不清楚。從結構的觀點推測，β-類澱粉胜肽與細胞膜的交互作用可能會改變β-類澱粉胜肽的結構，進而影響其聚集特性。本研究之主要目的是探討細胞膜在β-類澱粉胜肽聚集所扮演的角色，以溶血-肉豆蔻醯磷脂甘油(lyso-myristoylphosphatidylglycerol; LMPG)微胞(micelle)模擬細胞膜的環境及造成家族性阿茲海默症(familial Alzheimer’s disease)的荷蘭突變種β-類澱粉胜肽(Aβ40(E22Q))為模型，利用硫代黄素-T螢光光譜 (thioflavine-T fluorometric assay)、圓二色光譜學(circular dichroism spectroscopy)、電子顯微鏡(TEM)與核磁共振光譜學(nuclear magnetic resonance spectroscopy)等方法觀察細胞膜對β-類澱粉胜肽結構特性與聚集行為的影響。比較Aβ40(E22Q)在水與LMPG微胞環境下之結構與聚集行為得知，Aβ40(E22Q)在LMPG微胞環境下具有較高的α-螺旋成分(α-helical content)且其聚集速率被變慢。此現象意味了Aβ40(E22Q)與LMPG微胞的交互作用可增加其結構穩定度導致其不易聚集，這些結果可讓我們更進一步了解脂質與
β-類澱粉胜肽的交互作用在聚集過程可能所扮演的角色。

Alzheimer's disease (AD) a chronic neurodegenerative disease. It is the main cause of dementia in the elderly people. β-amyloid peptide (Aβ) is the main component of neuritic plaques which are a pathological hallmark of AD. β-amyloid peptide (Aβ) contains 39~42 amino acid residues. It was a proteolytic product derived from β-amyloid precursor protein (βAPP). Currently, the leading theory for explaining the etiology and pathogenesis of AD is the “Amyloid cascade hypothesis” which stated that Aβ aggregation resulted in brain cell death and dementia. Studies have shown that the aggregation of Aβ was linked to its interaction with cellular membranes. However, the underlying mechanism remains unclear. From a structural perspective, the interaction might induce conformational changes of Aβ resulting in an alteration of it’s aggregation behavior. To understand the role of cellular membranes in Aβ aggregation, the effects of lipids on the structure and aggregation behavior of Aβ were characterized by using thioflavine-T (Th-T) fluorometric assay, nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopies. An anionic lipid, lyso-myristoylphosphatidylglycerol (LMPG) and a genetic Aβ mutant, Dutch-type Aβ40 (Aβ40(E22Q)), were used in the present study. Structural studies indicated that LMPG micelles increased the α-helical content of Dutch-type Aβ40. The results of aggregation kinetics showed that LMPG micelles inhibited the aggregation of Dutch-type Aβ40. These results suggested that the interaction of Dutch-type Aβ40 with anionic lipids would increase its conformational stability leading to a reduction of its aggregation rate. These findings may help us gain an insight into the possible role of Aβ-lipid interaction in the process of Aβ aggregation.

目錄
誌謝 I
中文摘要 II
ABSTRACT III
目錄 IV
圖目錄 VI
表目錄 VIII
第一章 緒論 1
1-1 阿茲海默症(ALZHEIMER'S DISEASE) 2
1-1-1 阿茲海默症的病理特徵 2
1-1-2 類澱粉斑塊(Amyloid plaques) 2
1-1-3 神經纖維糾結物(Neurofibrillary tangles) 3
1-2 阿茲海默症的分類 (Classificaton of Alzheimer’s disease) 3
1-2-1 偶發型阿茲海默症(Sporadic Alzheimer’s disease；SAD) 4
1-2-2 家族型阿茲海默症(Familial Alzheimer’s disease；FAD) 4
1-3 類澱粉胜肽連鎖反應假說(AMYLOID CASCADE HYPOTHESIS) 6
1-4 -類澱粉胜肽之聚集 7
1-4-1 Aβ 7
1-4-2野生型Aβ 7
1-4-2 突變型Aβ 8
1-5利用細胞與小鼠模型研究類澱粉蛋白的影響 9
1-6以結構的觀點探討類澱粉蛋白之聚集 10
1-7阿茲海默症之診斷 11
1-7-1阿茲海默症之治療 11
1-8研究目的與計設 12
第二章 實驗材料與方法 14
2-1 實驗藥品 14
2-1-1 實驗儀器 15
2-2 樣品製備 16
2-2-1 質體建構(Plasmid construction)與轉型作用(Transformation) 16
2-2-2 Yeast ubiquitin hydrolase-1(YUH-1)的製備 18
2-2-3 A40(E22Q)的製備 19
2-3 圓二色光譜分析 21
2-4 TH-T螢光光譜分析 (THIOFLAVIN T FLUORESCENCE SPECTROSCOPY) 22
2-5 穿透式電子顯微鏡 (TRANSMISSION ELECTRON MICROSCOPY, TEM) 23
2-6 核磁共振光譜 (NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY) 24
第三章 結果 26
3-1 樣品的製備 26
3-1-1 YUH-1的製備 26
3-1-2突變型H6Ub-A40(E22Q)β-類澱粉胜肽的製備 28
3-1-3 Lyso-myristoylphosphatidylglycerol與Lyso-myristoylphosphatidylcholine微胞的製備 33
3-2 探討AΒ40(E22Q)與LMPG的交互作用 34
3-3探討LMPG對於A40(E22Q)的二級結構影響 41
3-3-1比較 A40(E22Q)在有無LMPG環境時二級結構之差異性 41
3-4探討A40(E22Q)的聚集機制在有無LMPG環境下有何差異 43
3-4-1利用Th-T螢光光譜分析A40(E22Q)的聚集機制在有無LMPG環境下有何差異 43
3-4-2利用圓二色光譜探討A40(E22Q)的聚集機制在有無LMPG環境下有何差異 46
3-4-3利用穿透式電子顯微鏡觀察A40(E22Q)的聚集機制在水與LMPG環境下比較形成纖維之速率 49
3-4-2 利用核磁共振光譜圖探討A40(E22Q)與LMPG脂質的交互作用機制 50
第四章 結論與討論 53
4-1 LMPG脂質與A40(E22Q)的交互作用 53
4-2 L17A/F19A突變能否增加A40(E22Q)在LMPG與LMPC環境下之結構穩定度 54
參考文獻 60

 
圖目錄
圖 1-1-1 β-類澱粉前驅蛋白(APP)水解途徑及家族性阿茲海默症突變位置……………P5
圖 2-2-1 His6-ubiquitin表現載體之建立…………………….…………………………..P17
圖 2-2-2酵母菌泛素水解酶(YUH-1)之純化流程……………………………………….P18
圖 2-2-3融合蛋白A40(E22Q)純化流程圖………………………………………………P20
圖3-1-1表達酵母菌泛素水解酶(Yeast Ubiquitin Hydrolase；YUH-1)之SDS-PAGZ...P26
圖 3-1-2 YUH-1經鎳親和性管柱層析(Immobilized Metal Affinity Chromatography)純化後以15% SDS-PAGE分析之電泳圖…………………………………………………..P27
圖 3-1-3表達β-類澱粉胜肽融合蛋白His6-ubiquitin- A40(E22Q)之SDS-PAGE ……P28
圖 3-1-4 His6-ubiquitin-A40(E22Q)合蛋白經鎳親和性管柱層析純化後以15% SDS-PAGE分析之電泳圖………………………………………………………………...P29
圖3-1-5利用His6-YUH-1水解作用切除H6Ub-A40(E22Q)之Tricine PAGE………...P30
圖3-1-6鹼性溶液純化A40(E22Q)之HPLC層析圖…………………………………...P30
圖3-1-7酸性溶液純化A40(E22Q)之HPLC層析圖…………………………………….P31
圖3-2-8 H6Ub-A40(E22Q)水解後之15% Tricine-SDS-PAGE…………………………P32
圖3-1-9 A40(E22Q)之質譜……………………………………………………………….P32
圖3-2-1 Aβ40(E22Q)在水環境以及LMPG微胞環境下整體二級結構差異性 ………..P34
圖3-2-2 Aβ40(E22Q)在水環境以及LMPG微胞環境下整體二級結構差異性…………P35
圖3-2-3 Aβ40(E22Q)在水環境以及LMPC環境下整體二級結構差異性……………….P36
圖3-2-4 Aβ40(E22Q)在水環境以及LMPC微胞環境下整體二級結構差異性 ……...…P37
圖3-2-5 A40(E22Q)在水環境下與LMPG微胞環境下之1H-HSQC光譜圖…………..P38
圖3-2-6 A40(E22Q)在水環境下與LMPC微胞環境下之1H-HSQC光譜圖…………..P39
圖3-2-7 A40(E22Q)在LMPG微胞與LMPC微胞環境下之1H-HSQC光譜圖……….P40
圖3-4-1 A40(E22Q)在水與LMPG微胞環境下時ThT螢光光譜分析…………………P43
圖3-4-2 A40(E22Q)在水與LMPG微胞環境下時ThT螢光光譜分析…………………P44
圖3-4-3綜合比較A40(E22Q)在水、LMPG、LMPC環境ThT螢光光譜分析…………P45
圖3-4-4 A40(E22Q)在水環境中的二級結構變化……………………………………….P46
圖3-4-5 A40(E22Q)在LMPG微胞環境中的二級結構變化 …………………………..P47
圖3-4-6 A40(E22Q)在LMPC微胞環境中的二級結構變化……………………………P48
圖3-4-7 A40(E22Q)在水環境下與LMPG環境下纖維形成速率比較………………....P49
圖3-4-8比較A40(E22Q)在水環境與LMPG環境下時13C化學位移數值差異………P50
圖3-4-9推測A40(E22Q)與LMPG交互作用機制………………………………………P52
圖4-2-1圖4-2-1 A40(L17A/F19A/E22Q)在水、LMPG、LMPC環境下整體二級結構……….P55
圖4-2-2比較A40(E22Q)與A40(L17A/F19A/E22Q)在LMPG下整體二級結構……..P56
圖4-2-3比較A40(E22Q)與A40(L17A/F19A/E22Q)在LMPC下整體二級結構……..P57
圖4-2-4 A40(L17A/F19A/E22Q)在水與脂質環境下之ThT螢光光譜圖……………...P58
圖4-2-5 A40(E22Q)與 A40(L17A/F19A/E22Q)在水，LMPG與LMPC環境下之ThT螢光光譜…………………………………………………………………………………...P59

表目錄
表2-2-1高效液相層析(HPLC)移動相程式………………………………………………P21
表 2-3-1遠紫外光圓二色光譜之蛋白質二級結構之波長特徵…………………………P22
表 2-6-1二十種胺基酸之H、13C、13C與13C'二級結構傾向化學位移(chemical shift)參考值……………………………………………………………………………………...P25
表3-1-1 Lyso-myristoylphosphatidylglycerol(LMPG)相關數值……………………….P33
表3-1-2 Lyso-myristoylphosphatidylcholine(LMPC)相關數值………………………...P33
表 3-3-1 A40(E22Q)在50 mM LMPG與LMPC，298 K，pH 7.2下之二級結構分析...P41
表 3-3-2 A40(E22Q)在50 mM LMPG與LMPC，310K，pH7.2下之二級結構分析….P42
表 3-4-1 A40(E22Q)在200 mM LMPG 、pH 7.2、298 K、50 mM磷酸鉀緩衝溶液中骨架原子化學位移………………………………………………………………………...P51
表 4-2-1 A40(L17A/F19A/E22Q)在水、LMPG、LMPC環境下整體二級結構特性傾向…………………………………………………………………………………………...P55

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