臺灣博碩士論文加值系統

阿茲海默症是一種由於類澱粉胜肽（Amyloid β-peptide, Aβ）在大腦血管壁上沉澱形成斑塊而造成神經細胞的死亡的連續性腦退化的疾病。一般而言，可以將阿茲海默症分為早發型與晚發型兩大類。目前發現的病例中，所有家族性遺傳的阿茲海默症都是早發型的，由類澱粉胜肽突變引起的阿茲海默症即是屬於這一類。類澱粉胜肽是由β分泌酶與γ分泌酶切割類澱粉胜肽前驅蛋白所產生的一段39-43個胺基酸組成的胜肽。從阿茲海默症患者的身上發現了有釵h不同的突變發生在類澱粉胜肽前驅蛋白之上，而這些突變剛好可以分為三大類，分別坐落於ㄓ尷c酶、β分泌酶與γ分泌酶的切點附近。一般認為發生在β分泌酶與γ分泌酶切點附近的突變會增加β分泌酶與γ分泌酶對類澱粉胜肽前驅蛋白的切割速率，因而促使類澱粉胜肽的生成。相反的，在ㄓ尷c酶切點附近的突變反而降低了ㄓ尷c酶的活性，相對性提高了β分泌酶與γ分泌酶的活性，因而使類澱粉胜肽產量增加。然而，有另一種解釋可以說明為何β分泌酶切點附近的突變會引發早發性阿茲海默症：這些突變可能影響類澱粉胜肽結構的改變，因而增加了類澱粉胜肽聚集(aggregation)的速度。我們的目標就是希望藉由研究這些突變種類澱粉胜肽的結構去探討這些突變是否在聚集的分子機制上扮演重要的角色。在本研究中，我們成左獄s備出含有野生種及突變種類澱粉胜肽基因的菌株，並且成左漯竁F及純化出同位素標定的類澱粉胜肽。這些同位素標定的類澱粉胜肽將提供作為核磁共振光譜學之結構分析用。我們相信了解野生種及突變種類澱粉胜肽結構上的差異可以提供一個解釋類澱粉胜肽聚集所誘發的阿茲海默症的機制。

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the progressive deposition of the aggregated amyloid β-peptide (Aβ) on the cell surfaces and in the walls of cerebral blood vessels. Aβ is a 39-43 amino acid polypeptide derived from proteolytic cleavages of the β-amyloid precursor protein (APP) by β- and γ-secretase. There are three groups of mutation sites in APP found in AD patients. These mutation sites locate near the ?, β- and γ-secretase cutting sites. It is believed that mutations near the β- and γ-secretase cutting sites induce an increase in cutting rates by both secretases, in turn enhancing the formation rate of Aβ. On the contrary, mutations near ?secretase cutting sites may result in a decrease in the cutting rate of ?secretase, but relatively increasing the cutting rate of β-secretase, in turn leading to an increase of Aβ formation. However, there is another explanation for why these mutants cause the early-onset of AD. Mutations near β-secretase cutting site may affect the conformational change of Aβ peptide, resulting in an increase in their self-aggregation. Thus, we aimed to analyze the structure of mutants that may play important role in the molecular mechanism of aggregation. In this study, we successfully cloned, cultured and purified the isotope labeled wild-type and mutants Aβ peptides. The purified peptides were subjected to analysis by NMR. We believe understanding the structural and conformational differences between wild-type and mutants of Aβ peptides will shed light on the mechanism of aggregated Aβ-induced AD.

圖目錄
1. 斑塊(plaque)和纖維(tangle)病理位置示意圖
2. APP結構示意圖
3. 由APP產生Aβ的過程.
4. Aβ fibrillogenesis模式示意圖
5. Aβ結構變化假說圖
6. 引子設計圖
7. 定序比對之結果
8. Aβ融合蛋白於M9培養基中表達的結果
9. 以LB broth與M9 medium 測試Aβ表達的情形
10. 以Ni2+ column純化Aβ融合蛋白之結果
11. 野生型 Aβ胜肽經逆相高效能液相層析儀（Reverse-phase
HPLC）純化後的結果
12. Arctic Aβ胜肽經逆相高效能液相層析儀（Reverse-phase HPLC）
純化後的結果
13. Flemish Aβ胜肽經逆相高效能液相層析儀（Reverse-phase HPLC）
純化後的結果
14. Iowa type Aβ胜肽經逆相高效能液相層析儀（Reverse-phase
HPLC）純化後的結果
15. Aβ (L17A, F19A)胜肽經逆相高效能液相層析儀（Reverse-phase
HPLC）純化後的結果
16. Aβ (L17A, F19A)質譜圖
17. Wild type Aβ 的核磁共振光譜2D-1H, 15N HSQC以及部分3D-1H,
15N, 13C HNCA分析結果
18. Arctic type Aβ 的核磁共振光譜2D-1H, 15N HSQC以及部分
3D-1H, 15N, 13C HNCA分析結果
19. Flemish type Aβ 的核磁共振光譜2D-1H, 15N HSQC以及部分
3D-1H, 15N, 13C HNCA分析結果
20. Aβ (L17A, F19A)的核磁共振光譜2D-1H, 15N HSQC以及部分
3D-1H, 15N, 13C HNCA分析結果
21. Aβ 1-60與Ab 1-40胺基酸序列比較圗
22. Aβ 1-60胜肽經逆相高效能液相層析儀（Reverse-phase HPLC）
純化後的結果
23. Aβ 1-60 質譜圖
24. 以1公升之M9培養基表達經純化後的Ab融合蛋白質及Ab胜肽產
量表

表目錄
1. 英文摘要
2. 中文摘要
3. Introduction
實驗材料與方法
一. 實驗材料及方法
1. 實驗材料與藥品
2. 點突變反應
2.1野生型Ab質體製備
2.2引子設計
2.3 PCR反應
2.4 Transformation
3. Protein Expression
3.1 小量表達現測試及飽和菌液製備
3.2中量表達
3.3 大量表達
4. Protein purification
4.1 Prepare Ni2+ Column
4.2 French Press and Ni2+ Column purification
4.3 Concentration, Dialysis
4.4 HPLC purification
二. 實驗結果與討論
1. 野生型及突變型β-Amyloid質體序列比對
2.. Ab表達與純化及鑑定之結果
3. NMR結果及Aβ (L17A, F19A)及Aβ 1-60製備
4. Reference

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