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研究生:謝耀文
研究生(外文):Yao-wen Hsieh
論文名稱:丹參抗C型肝炎病毒成份的篩選與機制的探討
論文名稱(外文):Identification and mechanistic investigation of the Chinese herb Danshen active components with anti-HCV activity
指導教授:鄭如茜鄭如茜引用關係
指導教授(外文):Ju-Chien Cheng
學位類別:碩士
校院名稱:朝陽科技大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:54
中文關鍵詞:C型肝炎病毒丹參
外文關鍵詞:danshenhepatitis C virus
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C型肝炎病毒(HCV)感染會引起持續發炎反應、有機會形成肝硬化最後演變成肝癌,目前用於治療HCV感染的主要方法是單獨處理干擾素(IFN)或是長效型干擾素(PEG-IFN)結合抗病毒藥物Ribavirin進行治療,但是治療過程會產生強烈的副作用,因此尋找新的治療方法,是一個重要的研究方向,本研究探討丹參萃取物或是丹參天然化合物中是否有抑制HCV的可能,並進一步去探討可能的作用機制。
首先我們採用HCV subgenomics replicon細胞培養系統進行加藥抑制實驗,以西方墨點法偵測細胞中HCV病毒蛋白質表現量評估丹參可以抑制HCV蛋白質的表現,並進一步確定其藥物的作用機制可能在抑制RNA dependent RNA polymerase (RdRp)的活性。因此建立HCV RdRp的活性測試平台,以評估藥物抑制的可能性。結果顯示,除了丹參粗萃取物可以抑制HCV蛋白質的產生外,進一步分析丹參中的其他主要化合物,顯示隱丹參酮能降低HCV RNA的產生、抑制 HCV病毒蛋白質的表現,HCV RdRp活性結果顯示隱丹參酮能抑制HCV RdRp活性。結合目前臨床的藥物長效型干擾素(PEG-IFN)的結果顯示兩個藥物在抑制HCV的特性上呈現協同的結果。
丹參已廣泛使用於臨床上治療心血管疾病、並記載可以改善肝纖維化症狀,我們結果則顯示,丹參萃取物及其化合物具有抑制HCV 感染的能力。
HCV infection causes persistent inflammation, and may sequentially develop liver cirrhosis that could progress further to hepatoma. Interferon alone or pegylated interferon(PEG-IFN) combined with ribavirin is the current treatment strategy. It will induce strong side effects during treating processes. Therefore, it is considerable to develop a new strategy to prevent HCV infection and / or increase the successful rate for the therapy of HCV infection. The aim of this research is to find out the anti-HCV activity of Danshen and its compounds and the possible mechanisms will also be investigated.
The subgenomic replicon was used to evaluate the efficacy of anti-HCV activity. Western blot assay was performed and the data showed that HCV viral protein expression was inhibited by Danshen. Since the anti-HCV activity may cause by RdRp acitivity inhibition, an HCV RdRp activity platform was set up.
In addition to Danshen crude extract, the derived compound, cryptotanshinone, can suppress HCV RNA production, viral protein expression and HCV RdRp activity. The combination assay demonstrated that cryptotanshinone and PEG-IFN showed synergistic effects on viral replication.
Danshen is widely used in the coronary heart disease, and has been reported to exert antifibrotic effects in hepatic fibrosis. Our data imply that Danshen consists of active components with intrinsic inhibitory activity on HCV infection.
摘要(中文)..........................................................................................................Ⅰ
摘要(英文)……………………………………………………………………..Ⅱ
誌謝.....................................................................................................................Ⅲ
目錄.....................................................................................................................Ⅳ

目錄
第一章 文獻回顧……………………………………………………………….1
1.1 C型肝炎病毒……………………………………………………………….1
1.1.1 HCV歷史回顧……………………………………………………………1
1.1.2 HCV的基因體組成………………………………………………………1
1.1.3非結構性蛋白質NS5B的表現、純化與活性測試…………………………….3
1.1.4 HCV的病毒複製系統-HCV replicon cell culture system……….….......5
1.1.5臨床治療…………………………………………………………………6
1.2丹參………………………………………………………………………...6
1.2.1丹參簡介………………………………………………………………….6
1.2.2丹參的生物活性…………………………………………………………7
1.3研究目的與策略…………………………………………………………...8

第二章 材料與方法…………………………………………………………..10
2丹參及其萃取化合…………………………………..…….……………......10
2.2化學藥品與儀器………………….………………………………………..10
2.2.1化學藥品…………….…………………………………………….……..10
2.2.2儀器………………………………………………………………………11
2.3細胞培養…………………………………………………………………...11
2.4蛋白質定量.………………………………………………………………12
2.5蛋白質電泳…………………………………………………………….…12
2.6西方墨點法……………………………………………………………….13
2.7 HCV NS5B表現質體的構築 …………………………………………...14
2.8轉形作用………………….………………………………………………14
2.9 NS5B蛋白質誘導………………………………………………………..14
2.10 NS5B蛋白質的純化……………………………………………………15
2.11 NS5B蛋白質RdRp活性的分析……………………………………….16
2.12 統計分析方法…………………………………………………………..16
2.13 藥物加成試驗………………………………………………………….17

第三章 結果………………………………………………………………….18
3.1丹參粗萃取物和丹參化合物對replicon細胞的影響…………………..18
3.2 HCV NS5B蛋白質表現質體的構築與蛋白質的誘導………………….19
3.3 NS5B蛋白質的純化……………………………………………………...20
3.4 NS5B蛋白質的RdRp活性的測試………………………………………20
3.5隱丹參酮和長效型干擾素具協同作用…………………………………..21

第四章討論……………………………………………………………………23
4.1丹參和丹參化合物對HCV的抑制效用………………………………...23
4.2 HCV RdRp的活性測定…………………………………………………..24
4.3臨床藥物與隱丹參酮……………………………………………………..25

圖表…………………………………………………………………………….26
圖1. 丹參SFE粗萃取物對於replicon細胞的影響……………………........26
圖2. 丹參酮 IIA對於replicon細胞的影響………………………………....27
圖3. 隱丹參酮對於replicon細胞的影響…………………………….……...28
圖4. 隱丹參酮對於replicon細胞內subgenomic HCV RNA的影響............29
圖5. 有機溶劑的丹參粗萃取物對於replicon細胞的影響…………….…...30
圖6. 限制酶切割確認構築後的質體是否正確……….…………...………...31
圖7. 以IPTG進行誘導前、誘導後的比較…………………………….…...32
圖8. 親和性管柱進行NS5B蛋白質的純化……………………………..….33
圖9. 親和性管柱純化一次和純化兩次的活性比較………………….…......34
圖10. 一價離子對RdRp的影響…………………..……………..…………..35
圖11. 二價離子對RdRp的影響…………………………………….……….36
圖12. 以gliotoxin測試RdRp的活性是否可以被抑制………………….....37
圖13. 隱丹參酮抑制RdRp的活性………………………………….……….38
圖14. 隱丹參酮和長效干擾素對於Replicon細胞的抑制效果….…………39
圖15. 長效型干擾素和隱丹參酮的結合效果………………………………..40
圖16. 干擾素與隱丹參酮的協同作用………………………………………..41
表1. Affinity column純化HCV NS5B蛋白質的產量………………..………42

附圖、附表……………………………………………………………………..43
附圖1. 經由HCV基因體能轉譯出十個結構與非結構性蛋白質………….43
附圖2. 丹參中主要成份的化學成份………………………………………...44
附圖3. 丹參萃取流程…………………………………………………………45
附圖4. HCV NS5B表現質體構築流程圖………………………………....…46
附圖5. RdRp的活性測定原理…………………….....……………………….47
附表1. 不同溶劑萃取丹參能得到不同的組成成份………………..…...…..48
參考文獻……………………………………………………………………….49
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