建立抑制C型肝炎病毒複製能力之研究平台

To Establish the Platform for Inhibition of HCV RNA Replication

翁甄憶(Zhen-Yi Weng)

指導教授 ： 柯俊良

中山醫學大學/醫學院/醫學分子毒理學研究所/碩士(2006年)

https://doi.org/10.6834/CSMU.2006.00030

摘要

HCV是一種正向單股的RNA病毒，屬於Flaviviridae家族成員之一。目前全球約有1億7千萬人口為慢性感染帶原者。臨床報告中指出，約有80%受C 型肝炎病毒感染者會引起慢性肝炎 (chronic hepatitis)，最後發展至嚴重肝硬化 (cirrhosis)及肝癌 (hepatocellular cacinoma；HCC)。近年來為研究HCV複製機轉，發展出含HCV subgenomic RNA的人類肝癌細胞株 Huh7，由neomycin phosphotransferase抗藥基因及encephalomyocarditis (EMCV) IRES取代HCV結構蛋白基因，利用HCV 5’端非轉譯區為HCV internal ribosome entry segment (IRES)，驅動下游基因表現，可以進行高效率的病毒基因複製。我們利用此HCV- Huh7 (HCV replicon subgenomic containing cells)細胞株來篩選20種藥物，希望找出具療效且較無副作用的抗HCV病毒藥物，並進一步探討其作用機制。首先，利用藥物處理HCV-Huh7細胞株後，以MTS assay、細胞計數、細胞型態，觀察藥物對細胞存活的影響及對細胞特異性毒殺。在藥物不影響細胞生長存活的狀況下，以NS5b的RT-PCR及HCV Taqman Probe Real-Time PCR分析藥物抑制HCV RNA複製的能力。我們發現有雙鶴極品靈芝、高三帖靈芝、白蓮蕉、魚針草具抑制HCV RNA複製的功能，其中又以魚針草 (Anisomeles indica) 最為明顯。進一步探討藥物抑制HCV基因複製的作用機轉，由Amplex Red試藥分析顯示，藥物中所含的H2O2濃度不會影響細胞中HCV-RNA表現的情形。以Griess Reagent測試得知，細胞處理IFN-Alpha或魚針草後，其NO2-濃度沒有明顯的變化產生。在人類肝癌細胞株Huh7中，魚針草會造成細胞死亡，以西方點墨法觀察，魚針草會誘發Huh7大量表現Cox-2且抑制NF-kappa B表現。但在HCV- Huh7中，魚針草不會影響細胞存活，但會些微增加Cox-2及核蛋白中NF-kappa B表現量，且可誘發Erk磷酸化。但分別以BAY11-7085及PD98059抑制NF-kappa B及Erk磷酸化後，並未能回復HCV複製能力，所以魚針草和IFN-Alpha抑制HCV複製能力可能與NF-kappa B和Erk無關。以二維電泳分析處理藥物後，細胞內蛋白表現改變。將有效抑制HCV基因複製的魚針草複合物，利用電灑法質譜分析藥物所含成分之分子量。所以，由本實驗室建立的抗HCV病毒藥物工作平台發現魚針草為值得進一步研究的新藥。

關鍵字

C型肝炎

並列摘要

Hepatitis C virus (HCV) is a positive-sensed, single-stranded RNA virus of the Flaviviridae family. Currently, it is estimated that there are more than 170 million people who are infected by HCV worldwide. About 80% of HCV infection results in chronic infection that can lead to severe liver diseases such as cirrhosis and hepatocellular carcinoma. Recently , a synthetic HCV subgenomic RNA including the 5’-untranslated region (UTR) as an internal ribosomal entry site (IRES), the neomycin phosphotransferase gene (Neo) and the encephalomyocarditis virus as (EMCV) internal ribosome entry site (IRES), instead of the structural protein-encoding region, replicated efficiently in the cell line Huh7 (Huh7-HCV). The Huh7-HCV cells were treatmented with drugs at concentrations that did not induce cell death and examined by reverse transcription polymerase chain reaction and Real-Time PCR to measure the copy numbers of HCV subgenomic RNA. Therefore, we have established the platform to monitor the inhibition of HCV replication. Furthermore, to investigate the effects of twenty drugs on HCV subgenomic RNA synthesis, we applied methods such as MTS assay, cell-number counting and cell morphology observation to evaluate the cytotoxicity of individual drugs. There are four effective drugs that could significantly inhibit the synthesis of HCV-RNA, including Shuang Hor Supreme Lingzhi, Shuang Hor Lingzhi (Triterpenoids), Canna indica, and Anisomeles indica. Using Amplex Red reagent assay, the concentrations of H2O2 in medium containing drugs did not affect the replication of HCV-RNA. In Griess Reagent system, there is no significant change for the concentrations of NO2- in medium containing IFN-Alpha or Anisomeles indica. Interestingly, Anisomeles indica could induce cell death in Huh7 cells, but not in HCV-Huh7 cells. By western blotting, Anisomeles indica could induce Cox-2 and suppress NF-kappa B in Huh7 cells. Anisomeles indica could induce Cox-2, NF-kappa B nuclear translocation slightly and Erk phosphorylation in HCV-Huh7. Pretreatments of BAY11-7085 or PD98059 did not restore the inhibiton of HCV replication with IFN-Alpha and Anisomeles indica. Therefore, the suppression of HCV replication by Anisomeles indica is not related to NF-kappa B and phosphor-Erk pathway. By 2-DE analysis, it is revealed that the pattern of protein expression was changed after treatment with Anisomeles indica and IFN-Alpha. In our research, Anisomeles indica has made a signification inhibition of HCV replication. It is merited to develop new therapeutic drug for inhibition of HCV.

並列關鍵字

HCV

參考文獻

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