To Establish the Platform for Inhibition of HCV RNA Replication

• Main Authors: Zhen-Yi, 翁甄憶
• Other Authors: Jiunn-Liang Ko
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/54036488061459011974

碩士 === 中山醫學大學 === 醫學分子毒理學研究所 === 94 === 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.