臺灣博碩士論文加值系統

類澱粉beta蛋白質(Abeta)是一種生理上的胜肽，由類澱粉前驅蛋白(APP)經一系列代謝途徑所生成，而釋出胞外的Abeta會被許多的內切胜肽酶進行降解代謝，若在腦中的生成與代謝之失衡，則會導致聚集的產生，此為阿茲海默症的成因。阿茲海默症為一種神經退化性疾病，由此可見其發病機制中，Abeta的生成是個十分重要的因素。
對於治療阿茲海默症，清除新生成的Abeta胜肽是可行的策略之一，腦啡肽酶(NEP)被視為清除Abeta的重要內切胜肽酶。在此研究中，我們合成淬滅螢光之胜肽受質來偵測腦啡肽酶的活性，而此胜肽所採用的是Abeta上前七個胺基酸殘基。一螢光分子連接在胜肽的N端，其發出的螢光會被存在於C端半胱氨酸側鏈上的一淬滅分子所消滅。但當此胜肽受質被內切胜肽酶水解，便會放出螢光。而我們實驗的結果也顯示了此檢測系統對於腦啡肽酶(NEP)與胰島素降解酶(IDE)有極高的靈敏性，而對於其他的Abeta降解酶則呈現相當低的靈敏性。此外，基於我們的發現，一套利用細胞進行之檢測系統已建立成形，可用於篩選能夠促進人類神經瘤母細胞上腦啡肽酶產量的藥物。



關鍵字：類澱粉beta蛋白質、Abeta降解酶、腦啡肽酶。

Amyloid-b (Ab), a physiological peptide, which is produced from amyloid precursor protein (APP) by sequential cleavages, and released into the extracellular spaces. The released Ab undergoes proteolytic degradation by multiple endopeptidases. The imbalance between the production and catabolism of Ab in the brain results in the accumulation of Ab leading to Alzheimer’s disease. Alzheimer’s disease is a neurodegenerative disease, and Ab in Alzheimer’s disease plays a pivotal role in the pathogenesis of the disease.
Neprilysin (NEP) has been singled out as the most promising candidate in Ab cleaning in the therapeutics of Alzheimer’s disease. In this study, a quenched fluorogenic peptide substrate whose sequence was adopted from the first seven residues of the Ab peptide was synthesized to detect the activity of neprilysin. A fluorophore was attached to the C-terminal Cys residue but its fluorescence was quenched by the existence of a quencher linked to the N-terminus of the peptide. When this peptide substrate was degraded by an endopeptidase, the fluorescence lights up to serve as a sensitive detection system for endopeptidase. Our results showed that this assay system was extremely sensitive to NEP and insulin degrading enzyme (IDE) while inert or much less sensitive to other Ab-degrading enzymes. Moreover, based on our findings, a cell-based assay system was developed to high throughput screen chemicals which are able to enhance NEP and production in human SH-SY5Y cells.

Key words：Amyloid-b, Ab-degrading enzymes, neprilysin

目錄

摘要 I
Abstract II
目錄 III
圖目錄 VI
表目錄 IX
第一章 緒論 1
第二章 文獻回顧 3
2-1 阿茲海默症簡介 3
2-1-1 類澱粉前驅蛋白(amyloid precursor protein) 8
2-1-2 類澱粉蛋白質 10
2-1-3 阿茲海默症的治療策略 11
2-2 類澱粉貝塔蛋白降解酵素 16
2-2-1 Neprilysin 19
2-2-2 Insulin-degrading enzyme(IDE) 24
2-2-3 Endothelin-converting enzyme-1 (ECE-1) 25
2-2-4 Angiotensin-converting enzyme (ACE) 26
2-2-5 Plasmin 26
2-2-6 Matrix metalloproteinases (MMPs) 27
2-3 體抑素(Somatostatin) 29
2-4 固相胜肽合成法介紹 31
2-5螢光共振能量轉移(Fluorescence Resonance Energy Transfer, FRET) 34
2-6螢光探針分子簡介 37
2-6-1 Thiol-reactive dye 37
2-6-2 Alexa Fluor 350 C5-Maleimide 38
2-6-3 The Alexa Fluor Dye Series 40
2-6-4 Amine-reactive dye 41
2-6-5 4-((4-(dimethylamino)phenyl)azo)benzoic acid, succinimidyl ester (Dabcyl) 42
2-7 偵測NEP活性的方法 43
第三章 研究動機 49
第四章 實驗裝置、藥品與步驟 50
4-1 實驗藥品 50
4-1-1 水 50
4-1-2 化學藥品 50
4-2 實驗儀器 53
4-3 實驗步驟 54
4-3-1 類澱粉b蛋白質片段Ab (1-7)C的合成、純化與鑑定 54
4-3-2 胜肽A(1-7)C和螢光供體Alexa-350之結合 55
4-3-3 Alexa350-A(1-7)C和quencher之結合 56
4-3-4 qf-Ab(1-7)C之純化與身分鑑定 57
4-3-5 螢光光譜分析 57
4-3-6 溶劑濃度對酵素NEP與受質qf- Ab (1-7)C反應之螢光光譜分析 58
4-3-7 酵素NEP對受質qf-Ab(1-7)C螢光檢測之靈敏度 58
4-3-8 不同的類澱粉蛋白降解酵素對受質qf- Ab (1-7)C之螢光分析 59
4-3-9 利用Thiorphan探討酵素活性之抑制 60
4-3-10 SH-SY5Y細胞之培養 60
4-3-11 在細胞上進行受質qf- Ab (1-7)C之螢光分析 61
4-3-12 利用somatostatin處理細胞 62
第五章 結果與討論 64
5-1 胜肽受質qf- Ab (1-7)C之合成與身分鑑定 64
5-1-1 類澱粉貝塔蛋白片段Ab (1-7)C之合成與身分鑑定 65
5-1-2 Alexa-350- Ab (1-7)C之合成與身分鑑定 68
5-1-3 qf- Ab (1-7)C之合成與身分鑑定 74
5-2 探討溶劑濃度對酵素NEP與受質qf- Ab (1-7)C反應之影響 82
5-3 酵素NEP對受質qf- Ab (1-7)C螢光檢測之靈敏度探討 86
5-4 酵素專一性之檢測 90
5-5 利用Thiorphan探討酵素活性之抑制 92
5-6 在細胞上進行受質qf- Ab (1-7)C之螢光分析 95
5-7 利用somatostatin誘導類澱粉蛋白降解酵素之探討 97
5-8 不同濃度somatostatin誘導類澱粉蛋白降解酵素之比較 99
第六章 結論與建議 101
6-1 實驗結論 101
6-2 建議與未來展望 103
參考文獻 104

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