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研究生: 鄭詠馨
Jheng, Yong-Sin
論文名稱: 應用果蠅模式篩選中草藥抑肝散(順寧意)緩解阿茲海默氏症的醫療潛力
Application of Drosophila Model to Screen Medical Potential of the Chinese Herbal Medicine YIGANSAN (SHUNNINGYI) for Alleviating Alzheimer's Disease
指導教授: 吳忠信
Wu, Chung-Hsin
口試委員: 蘇銘燦 莊武璋
口試日期: 2021/07/30
學位類別: 碩士
Master
系所名稱: 生技醫藥產業碩士學位學程
Graduate Program of Biotechnology and Pharmaceutical Industries
論文出版年: 2021
畢業學年度: 109
語文別: 中文
論文頁數: 31
中文關鍵詞: 阿茲海默氏症Aβ毒性Tau毒性免疫磁減量技術果蠅
英文關鍵詞: Alzheimer's disease, Aβ toxicity, Tau toxicity, immunomagnetic reduction, Drosophila
研究方法: 實驗設計法
DOI URL: http://doi.org/10.6345/NTNU202101065
論文種類: 學術論文
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  • 傳統中藥抑肝散過去常用於治療神經退行性疾病、精神官能症、失眠。而阿茲海默氏症是失智症裡各類型中最常見的,主要是以澱粉樣蛋白β (Aβ) 的異常堆積和Tau蛋白的過度磷酸化造成腦部細胞損傷有關。本文利用DPPH檢測中藥抑肝散的自由基清除與抗氧化能力;再透過細胞存活率分析法(MTT assay)檢視中藥抑肝散對於SH-SY5Y神經細胞的保護作用,實驗結果顯示中藥抑肝散具有相當高的自由基清除與抗氧化能力,對於SH-SY5Y神經細胞也有顯著的保護作用。本文利用轉基因果蠅表現Aβ42和hTauR406W毒性蛋白質作為AD動物模式,研究中藥抑肝散對人類Aβ 和人類Tau蛋白過度累積所引發的神經毒性在果蠅上是否具有保護作用。由於人類Aβ蛋白過度表現的Aβ42 會產生複眼退化;因此利用綠螢光蛋白觀察Aβ42 表現所造成的複眼退化,以及計算hTauR406W的背甲剛毛數目,可以評估中藥抑肝散對於Aβ和Tau蛋白過度表現果蠅的神經保護作用,實驗結果顯示中藥抑肝散可以緩解Aβ42 複眼的退化;另外也可以顯著緩解hTauR406W果蠅的背甲剛毛退化。此外,透過高靈敏的免疫磁減量(ImmunoMagnetic Reduction,IMR)技術可以評估Aβ42的Aβ蛋白表現,以及評估hTauR406W的Tau蛋白表現,結果顯示中藥抑肝散可以降低Aβ蛋白以及Tau蛋白的表現。歸納研究結果,可以說明中藥抑肝散對於緩解阿茲海默氏症可能具有替代醫療效果。

    Yi-Gan-San (YGS), a traditional herbal medicine, has been used to manage neurodegenerative disorders and treat neurosis, insomnia, and dementia. Alzheimer’s disease (AD), a main cause of dementia, is the most common neurodegenerative disease that is related to the abnormal accumulation of amyloid β (Aβ) and Tau proteins. This master's thesis aims to use transgenic AD fly models overexpressing Aβ42 and Tau proteins to evaluate whether the traditional Chinese medicine Yigansan (YGS) exhibits a neuroprotective effect against the toxicity of accumulated Aβ and Tau proteins in Drosophila. In this study. 1,1-diphenyl-2-picrylhydrazyl (DPPH) was used to detect the free radical scavenging and anti-oxidant ability of YGS; then the cell viability (MTT) assay was used to examine the protective effect of YGS on SH-SY5Y neuroblastoma cells. Our results showed that YGS exhibited high free radical scavenging and antioxidant capacities and has a significant protective effect on SH-SY5Y cells. Next, we observed the green fluorescent protein expression of Aβ42 flies and calculated the number of bristles of hTauR406W flies to evaluate the neuroprotective effect of the YGS on the over-expression of Aβ42 and Tau toxicity in Drosophila. Our results showed that YGS can significantly alleviate degeneration but enhance green fluorescent protein expression in the eyes of Aβ42 flies; also, YGS can significantly alleviate bristles degradation in hTauR406W flies. In addition, through highly sensitive immunomagnetic reduction (IMR) technology, the Aβ42 and Tau protein expressions of Drosophila can be evaluated. Our results showed that YGS can significantly reduce the Aβ42 protein expression of Aβ42 flies and the Tau protein expression of hTauR406W flies. Our findings suggest that YGS may have a beneficial alternative therapy for AD via alleviating Aβ and Tau neurotoxicity that might help to alleviate the symptoms of neurodegeneration and dementia in the elderly.

    中文摘要 I ABSTRACT II 目錄 III 圖表次 V 第一章 緒論 1 第一節 研究背景 1 第二節 文獻探討 4 第三節 研究目的 5 第二章 材料與方法 6 第一節 實驗設計 6 第二節 果蠅基因型 7 第三節中草藥抑肝散的高效液相層析3D-HPLC圖譜分析8 第四節 細胞存活率分析法 (MTT assay) 8 第五節 DPPH氧化自由基能力測定 8 第六節 免疫磁減量(ImmunoMagnetic Reduction)技術 9 第七節 統計與資料分析 9 第三章 實驗結果 10 第一節 中草藥抑肝散的高效液相層析3D-HPLC圖譜 10 第二節 中草藥抑肝散的抗氧化清除自由基的能力 10 第三節 中草藥抑肝散的SH-SY5Y神經細胞保護作用 11 第四節 中草藥抑肝散緩解Aβ42蛋白毒性造成的果蠅複眼感光神經細胞退化 11 第五節 中草藥抑肝散緩解Gmr>Aβ42的Aβ42蛋白毒性表現 12 第六節 中草藥抑肝散緩解hTauR406W蛋白毒性造成的背甲剛毛神經退化 12 第七節 中草藥抑肝散緩解hTauR406W的Tau蛋白毒性表現 13 第四章 討 論 14 第一節 中草藥抑肝散的抗氧化與緩解細胞毒性功能 14 第二節 中草藥抑肝散透過抑制Aβ42蛋白毒性表現緩解果蠅複眼的神經性退化 15 第三節 中草藥抑肝散透過抑制hTauR406W蛋白毒性緩解果蠅背甲剛毛的神經性退化16 第四節 中草藥抑肝散應用在緩解阿茲海默氏症的醫療潛力 16 第五章 結 論 17 參考文獻 18 附錄圖表 25

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