小柴胡汤、复方黄芪、丙肝灵在体外对HCV结构区基因转录的抑制作用

Inhibitory Effect of Chinese Herbs Xiao Chai Hu Tang, Fu Fang Huang Qi and Bing Gan Ling on Transcription of Hepatitis C Virus Structural Genes in vitro

目的:评价丙肝灵、小柴胡汤、复方黄芪3种中药在体外对HCV结构区基因转录的抑制作用,探讨中药抗HCV作用的机制。方法:在能稳定表达HCV结构区基因的HelaD细胞培养基中,加入不同浓度小柴胡汤、复方黄芪、丙肝灵3种中药培养48小时后,通过MTT法和对RT-PCR的扩增片段进行扫描的半定量方法分别检测3种中药对HelaD细胞的细胞毒性最低浓度以及对HCV结构区基因转录量的变化。结果:①小柴胡汤、复方黄芪及丙肝灵在浓度分别为1g/ml、0.8g/ml、0.6g/ml、0.4g/ml时,HelaD细胞与不加药的正常组相比,其存活率都低于95％。(P<0.05)。②在3种中药浓度为0.1g/ml时,HelaD细胞经RT-PCR得到的DNA片段显示在紫外光下与内对照GAPDH基因DNA片段的亮度比值分别为:0.24、0.10和0.12;在3种中药浓度为0.001g/ml时,该比值则分别为0.75,0.67和0.61。结论:小柴胡汤、复方黄芪及丙肝灵在浓度低于0.2g/ml时对HelaD细胞已无毒性作用;在3种中药浓度分别为0.1g/ml,0.01g/ml,0.001g/ml时,对HCV结构区基因转录都有抑制作用,且随着药物浓度的减低,对HCV mRNA转录量均分别相应地减少。

Objective: In order to study the effects of Chinese herbs on Hepatitis C virus (HCV), Xiao Chai Hu Tang (XCHT), Fu Fang Huang Qi (FFHQ) and Bing Gan Ling (BGL) were selected to value their inhibition on transcription of hepatitis C virus structural genes in vitro respectively. Methods Several different concentrations of XCHT, GGHQ and BGL were respectively added to the medium for Hela D cells, which can express HCV structural genes, to make sure the lowest poison concentration to the HelaD cells, The lowest poisonous concentrations herbs were added to the HelaD cells medium and the total mRNA was used to identify the results of RT-PCR and the inner control gene GAPDH and then, the half-quantity of RNA were analyzed according to the fluorescent density after screened. Results: For XCHT, FFHQ and BGL, the lowest poisonous concentration to the Hela D cells was of 0. 2g/ml. Affer co - culturing of Hela D cells with the three herbs in the medium for 48 hours respectively, the transcription of HCV structural genes were decreased with the increase of the concentration of the three kinds of herbs, because the fluorescent density of RT-PCR product bands were descended after gel electropheresis were screened under gel screening system. The ratio of the lightness of the RT-PCR product bands of XCHT, FFHQ and BGL to GAPDH gene respectively were 0.24, 0.10, and 0.12 which of concentration were all of 0.1g/ml. When the concentrations of XCHT, FFHQ and BGL were of 0.001g/ml, the ratios were 0.75, 0.67, 0.61 in order. Conclusion: The XCHT, FFHQ and BGL can inhibit the transcription of HCV structural genes in the HelaD cells. HelaD cells can be used as a cell model to select the anti-HCV drugs.