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研究生: 黃鉦翔
Chen-Hsiang Huang
論文名稱: 以amyloid-β 聚集為目標的阿茲海默氏症治療策略
Therapeutic strategies targeting amyloid-β aggregation for Alzheimer's disease
指導教授: 李桂楨
Lee, Guey-Jen
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2013
畢業學年度: 101
語文別: 中文
論文頁數: 55
中文關鍵詞: 阿茲海默氏症老年癡呆症澱粉樣蛋白斑β-結構澱粉樣蛋白42
英文關鍵詞: Alzheimer's disease, dementia, amyloid plaques, cross-β-structure, Aβ42
論文種類: 學術論文
相關次數: 點閱:607下載:47
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  • 阿茲海默氏症是最常見的老年癡呆症,主要臨床症狀包括Aβ胜肽聚集而成的細胞外澱粉樣蛋白斑與tau蛋白形成的細胞內神經纖維糾結。Aβ胜肽與tau蛋白的聚集往往是基於β-結構互相堆疊而形成。由於藉由破壞蛋白之間的氫鍵可能抑制蛋白聚集,故可能可以從吲哚、多酚類及其衍生物和中藥材篩選出潛在的Aβ胜肽聚集抑制劑。為了達成以上目的,本實驗利用thioflavin T分析法篩選人工合成之化合物。此外,於細胞層面上將Aβ42胜肽與GFP螢光蛋白的N端融合,用於反映Aβ42聚集之程度,並建立於SH-SY5Y細胞和293細胞中,篩選出Tet-On SH-SY5Y細胞與Tet-On 293細胞。Tet-On 293細胞被用來測試植物萃取物與天然或人工合成化合物,藉由綠色螢光訊號區別可延緩或抑制Aβ42聚集之抑制劑。本實驗使用Tet-On 293阿茲海默氏症細胞模式於高通量分析系統,並結合自動顯微鏡與圖像分析自動測試化合物的有效濃度。本實驗分析出10種植物萃取物與人工合成化合物,能提升Aβ42-GFP綠螢光之訊號,其中NTNU-043、NTNU-057、NTNU-059、NTNU-071、NC009-1以及NC009-2等較具潛力,能增加Hsp27蛋白表現,幫助抑制Aβ42聚集。

    Alzheimer's disease is the most common form of dementia that is pathologically characterized by the presence of extracellular amyloid plaques formed from Aβ peptide and intracellular neurofibrillary tangles formed from tau protein. Aggregation is often based on the formation of cross-β-structure and may be inhibited by disrupting the hydrogen bonds between sheets. Thus screening for indole, polyphenol derivatives and herbal medicines might find out potential inhibitors of Aβ peptide aggregation. To do this, thioflavin T assay was used to screen the synthetic compounds. Also, Aβ42 was fused to the N-terminus of GFP to couple the aggregation state with the fluorescence of GFP and used to generate Tet-On SH-SY5Y cell and 293 cell clones. Tet-On 293 cells were used to screen herbal extracts and natural or synthetic compounds. Inhibitors that retard or block Aβ42 aggregation can be distinguished by the increasing green fluorescent signal. The effective concentration of the tested herbs and compounds will be determined by using a high content analysis system that combines automated microscopy and automated image analysis in 293 AD model. In this study, 10 herbal extracts and synthetic compounds which could significantly increase green fluorescent signal were identified. Among the 10 compounds examined, NTNU-043, NTNU-057, NTNU-059, NTNU-071, NC009-1 and NC009-2 effectively increased green fluorescent signal and enhanced Hsp27 expression.

    目錄 I 中文摘要 III Abstract IV 壹、緒論 1 一、阿茲海默氏症 1 二、β-amyloid 蛋白 2 二、藥物篩檢之方向與模式 3 貳、研究目的 6 參、研究材料與方法 7 一、pET-32b(+)-Trx-His6-Aβ42、pcDNA5/FRT/TO/Aβ42GFP重組質體之構築 7 (一) pET-32b(+)-Trx-His6-Aβ42重組質體 7 (二) pcDNA5/FRT/TO/Aβ42-GFP重組質體 7 二、重組質體的選殖 8 (一)轉型勝任細胞的製備與保存 8 (二) DNA片段純化與接合反應 9 (三)細菌電穿孔轉型作用 9 (四)質體DNA小量製備 10 (五)質體DNA大量製備 11 三、融合蛋白於細菌中表現與純化 12 四、Thioflavin T螢光分析 13 五、細胞培養、轉染與分析 14 (一)細胞株繼代培養 14 (二)基因轉染 15 (三)細胞影像分析 15 (四)蛋白質萃取與西方轉漬分析 16 (二) RNA萃取 18 (三)反轉錄PCR 18 (四)同步定量PCR 19 六、Aβ42-GFP細胞株之候選藥物篩檢 19 肆、結果 21 一、Trx-His6-Aβ42重組質體的確認 21 二、Trx-His6-Aβ42蛋白的表現 21 三、Thioflavin T螢光分析Trx-His6-Aβ42蛋白聚集情況 21 四、Doxycycline誘導表現Aβ42-GFP細胞之建立 23 五、誘導表現Aβ42-GFP細胞株之藥物篩檢 24 六、候選藥物處理Aβ42-GFP細胞株活化Chaperone之情況 25 伍、討論 27 一、以Thioflavin T 螢光分析藥物對Trx-His6-Aβ42蛋白的影響 27 二、誘導表現Aβ42-GFP細胞株建立 27 三、以藥物結構、抗氧化與Chaperones探討藥物與疾病的關係 29 四、誘導表現Aβ42-GFP細胞株之藥物篩檢 30 五、候選藥物處理與Chaperone之活化 32 六、候選藥物處理與Aβ42-GFP表現 33 陸、參考文獻 34 柒、附錄圖表 43

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