氧化苦参碱抑制体外细胞培养模型HCV感染靶细胞研究

Oxymatrine inhibits target cell infection in the HCVcc system

目的通过构建HCV体外细胞培养模型（HCVcc）系统，验证氧化苦参碱抗HCV的作用。方法通过构建HCVcc系统，检测氧化苦参碱对感染HCV靶细胞24、48h和72h细胞增殖的影响和氧化苦参碱组和对照组中感染HCV靶细胞中HCVmRNA的表达水平变化，免疫荧光观察氧化苦参碱对Huh7．5．1细胞HCV蛋白荧光表达情况，采用单因素方差分析和单样本t检验判定统计学意义。结果2、4、8和12g／L氧化苦参碱分别作用于感染J6cc的Huh7．5．1细胞24、48、72h后，即呈明显的时间一剂量依赖性（P〈0．05）；氧化苦参碱处理组中感染J6cc的Huh7．5．1细胞中HCVmRNA相对表达量为0．59±0．12，与正常对照组基数1相比，HCVmRNA表达水平明显下降（t=8．909，P〈0．05）；随着氧化苦参碱药物浓度2～12g／L逐渐增大，HCV蛋白荧光表达量逐渐减弱。结论成功构建J6ccHCV系统，初步验证了氧化苦参碱的抗丙型肝炎病毒作用。

Objective To validate the antiviral effect of oxymatrine against hepatitis C virus （HCV） in the construction of the cell culture-based infectious virus system HCVcc. Methods An HCVcc system was established by infecting Huh7.5.1 cells with the J6 adapted virus （J6cc）. Cells of the HCVcc system were then treated with different concentrations of oxymatrine for 24 h, 48 h and 72 h, or left untreated （controls）. MTT assay was used to detect effects on proliferation. Real-time quantitative PCR was used to detect effects on HCV mRNA expression level, and immunofluorescence was used to detect effects on HCV protein expression. Data were expressed as mean±standard deviation, and statistically assessed using one-way ANOVA and one-sample t-test. Results Treatment with 2, 4, 8 and 12 mg/mL of oxymatrine for 24 h inhibited proliferation of the J6cc- infected cells by 8.4%, 15.2%, 29.6% and 48.6% respectively, 48 h treatment inhibited by 14.3%, 25.7%, 46.1% and 66.4% respectively, and 72 h treatment inhibited by 36.5%, 46.6%, 70.6% and 85.4% respectively. Thus, the effects of oxymatrine were time- and dose-dependent （P 〈 0.05）. The mRNA expression level in the oxymatrine- treated cells of the HCVcc system was 0.59 ~ 0.12, which was significantly lower than that in the control cells （P 〈 0.05）. Moreover, as the oxymatrine concentration increSed from 2 mg/mL to 12 mg/mL, the expression levels of HCV proteins also showed a decreasing trend. Conclusion We successfully constructed a J6cc infection HCVcc system and verified the antiviral effect of oxymatrine against HCV.