环孢素A抑制丙型肝炎病毒JFH-1复制的实验研究

Effects of Cyclosporine A on Hepatitis C Virus Replication in Human Hepatocytes

目的探讨环孢素A(cyclosporine A,CsA)对全基因组丙型肝炎病毒JFH-1(hepatitis C virus,HCVJFH-1)在肝细胞中复制的影响。方法以体外转录的HCVJFH-1RNA转染Huh7细胞,制备含HCV病毒颗粒的感染上清,建立全基因组HCVJFH-1感染Huh7细胞模型,分为对照组、CsA处理组,分别予以不同浓度、不同时间、不同时机和方法的CsA处理。通过实时荧光定量聚合酶链反应(real ti me polymerase chain reaction,RT-PCR)检测CsA对HCVJFH-1在Huh7细胞中复制的影响。结果与对照组比较,CsA(0.15、0.3、0.6、1.2μg/ml)处理组Huh7细胞中HCVJFH-1RNA相对表达量分别下降为(45.7±6.5)%、(18.9±2.1)%、(4.6±0.7)%、(2.1±0.2)%,上清中HCV拷贝数(×107/ml)较对照组(29.70±0.15)下降为(0.80±0.19)、(0.44±0.07)、(0.29±0.07)、(0.17±0.06)。CsA处理组HCV蛋白合成、上清中病毒颗粒的含量及Huh7细胞裂解液和上清感染力明显下降。同时CsA预处理较感染同时及感染后处理可更显著抑制HCVJFH-1在Huh7细胞中的复制,而撤药则导致其复制的反弹。以上差异均有统计学意义。结论 CsA可有效抑制全基因组HCVJFH-1在Huh7细胞中的复制及合成,并减少有感染力的HCV病毒颗粒产生。

Objective To investigate the effect of cyclosporine A（CsA） on full cycle hepatitis C virus（HCV） JFH-1 replication in human hepatocytes.Methods Infectious HCV JFH-1 generated in Huh7 cells by transfection of in vitro transcribed genomic JFH1 RNA was used to infect naive Huh7 cells.The infected cells were treated by CsA with different concentrations,time courses and strategies.HCV replication was determined by real time polymerase chain reaction（RT-PCR）.Results After CsA（0.15,0.3,0.6,1.2 μg/ml） treatment,the relative HCV JFH-1 RNA expression compared with the control group decreased to（45.7±6.5）%,（18.9±2.1）%,（4.6±0.7）%,（2.1±0.2%） and HCV copies（×10^7/ml） in the supernatant decreased to（0.80±0.19）、（0.44±0.07）,（0.29±0.07）,（0.17±0.06） respectively.In the CsA group,the HCV protein synthesis,virus contents in the supernatant,and the infectivity of Hun7 cell lysis solution and supernatant were significant decreased.Furthermore,CsA preprocessing inhibited the HCV JFH-1 better than CsA treatment during and after infection,and CsA withdrawal resulted in significant virus replication reoccurrence.Conclusion CsA can effectively inhibit the full cycle HCV JFH-1 replication in Huh7 cells and reduce production of infectious HCV virus.