https://scholars.lib.ntu.edu.tw/handle/123456789/159881
標題: | 於C型肝炎病毒感染之細胞中使修飾過的caspase3其細胞凋亡活性最佳化之研究 Optimizing apoptotic activity of a modified caspase3 in cells infected by Hepatitis C virus |
作者: | 張吉智 Chang, Chi-Chih |
關鍵字: | C型肝炎;Hepatitis C virus | 公開日期: | 2005 | 摘要: | 我們採用一種新的策略來發展抗C型肝炎病毒( Hepatitis C virus )的療法。針對C肝病毒的NS3蛋白酶,修改pro-apoptotic分子使它能被病毒的蛋白酶所活化,然後引起細胞凋亡(apoptosis)來清除受感染的細胞。我們選擇Cofilin 和 Caspase3為目標,然後依據病毒蛋白酶之專一性來做修飾。在Cofilin 與 Caspase3上導入各式各樣能被病毒NS3蛋白酶所辨認的胜肽序列 (NS3/4A, NS4A/4B, NS5A/5B),我們產生了許多包含這些修飾分子的質體(plasmid),然後在一個體外模擬系統中來測試它們 ─ 把含有病毒NS3蛋白酶的質體(plasmid)與修飾分子的質體一起轉殖(transfection)到293T細胞中。 那些包含NS5A/5B 或者NS4A/4B 胜肽序列之Cofilin能被病毒的NS3蛋白酶切開,然而不幸地,在處理過後它們不能在293T中引起細胞凋亡。 某些修飾過的caspase3能夠被病毒的NS3蛋白酶所活化,在運用西方墨點法(western blotting)分析蛋白表現量以及細胞凋亡的標誌之後,F88(caspase3 -NS5A/5B-88) 和 M (caspase3-modified 2)兩者脫穎而出。我們運用流式細胞技術(Flow cytometry)來定量分析它們細胞凋亡的程度,藉此探討轉殖劑量、效力強度和毒性之間的關係。分析之後,F88與M皆能有效地引發細胞凋亡,然而高劑量F88卻會出現一些毒性反應。 我們進一步於 HCV subgenomic replicon (HCV type 1b NS3-NS5B ) 中測試M (caspase3-modifed 2)的效力。它能誘導一小部分的細胞產生細胞凋亡,然而整體效果卻不夠顯著。原因主要是低落轉殖的效率或者在replicon中,只有一小部分的修飾分子被病毒蛋白酶所作用。 To develop anti- Hepatitis C virus therapy, we take a new strategy through modifying the pro-apoptotic molecule which could be specific activated by virus NS3 serine protease and then eliminating infected cells by apoptosis. Cofilin and caspase3 are selected as our targets and modified according to the specificity of HVC NS3 protease. Several plasmids encoding modified Cofilin and modified Caspase3 have been generated through introducing different peptide sequence recognized by HCV NS3 protease (NS3/4A, NS4A/4B, NS5A/5B site)(figure2,3). They are further examined on in-vitro mimetic system through co-transfecting with HCV NS3 protease into 293T cell. Some modified cofilin which carry NS5A/5B site or NS4A/4B site could be cleaved by HCV NS3/4A protease, but they failed to induce apoptosis in 293T cell after processing. Some modified caspase3 could be activated by HCV NS3 protease and induce cell apoptosis. After analyzing the protein expression level and the apoptosis makers through western immunoblotting, two clones - F88 (caspase3-NS5A/5B-88) and M (Caspase3-modified 2) were selected as our candidates. We examined the relationship between transfection dose, potency and toxicity by quantitating the degree of apoptosis with flow cytometry. After analysis, both clone could induce apoptosis efficiently. However, F88 clone showed certain extent of toxicity with higher transfection dose. M clone (caspase3-modifed 2) was further examined in HCV subgenomic replicon (HCV type 1b NS3-NS5B in Huh7).It induced apoptosis at small population of cells, but the apoptotic effect was not significant. The reason may be low transfection efficiency and/or low efficient cleavage by NS3/4A protease in HCV replicon. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/51352 | 其他識別: | en-US |
顯示於: | 分子醫學研究所 |
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ntu-94-R91448007-1.pdf | 23.31 kB | Adobe PDF | 檢視/開啟 |
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