【摘要】 目的:探讨siRNA能否有效抑制丙型肝炎病毒(HCV)感染细胞中HCV的复制。方法:用HCV JC1质粒体外制备HCV RNA转录体,转染Huh-7.5.1细胞,收集细胞上清,再次孵育Huh-7.5.1细胞以建立HCV感染细胞模型,并用间接免疫荧光检测细胞内HCV核心蛋白的表达。将针对HCV NS5B基因的siRNA转染HCV感染细胞(浓度为4、40和200 nmol/L,时间为24 h、48 h和72 h),将4、40和200 nmol/L siRNA转染正常Huh-7.5.1细胞,并设空白对照、脂质体对照、无关siRNA对照以及IFNα-2b(1 000 IU/ml)组,用荧光定量PCR检测细胞内HCV RNA和PKRmRNA水平。结果:HCV感染的Huh-7.5.1细胞中检测出HCV核心蛋白表达。40 nmol和200nmol/L siRNA 24 h组及4、40、200 nmol/L siRNA 48 h和72 h组,HCV RNA水平分别降至58%、54%、29%、17%、17%、33%、22%、19%,明显低于对照组(P<0.05或P<0.01)。1 000 IU/mlIFN的各时间组HCV RNA水平均明显低于对照组(P<0.05或P<0.01)。各浓度siRNA组的PKR mRNA的表达与对照组无明显差异(P>0.05)。结论:针对HCV NS5B区的siRNA能够明显抑制HCV感染细胞中HCV的复制,而不引起双链RNA依赖的蛋白激酶(PKR)基因表达的激活。更多还原

【Abstract】 Objective: To investigate the inhibitive effects of small interfering RNA(siRNA) on hepatitis C virus(HCV) replication in cells infected by HCV in vitro. Methods: The HCV RNA transcripts prepared by pFL-JC1 were transfected into Huh-7.5.1 cells.Na ve Huh-7.5.1 cells were incubated with the supernatants of transfected cells and the expression of HCV core protein in infected cells was detected by indirect immunofluorescence.The infected cells were transfected with 4,40 and 200 nmol/L of NS5B siRNA for 24 h,48 h and 72 h,respectively.The normal Huh-7.5.1 cells were transfected with 4,40 and 200 nmol/L of NS5B siRNA.Group of blank,lipofectamine 2000,unrelated siRNA and IFNα-2b(1 000 IU/ml) served as controls.The HCV RNA and PKR mRNA levels were examined by quantitative RT-PCR. Results: The HCV core protein in HCV infected cells was detected.Compared with control groups,the HCV RNA levels in infected cells significantly decreased when transfected with 40 and 200 nmol/L of siRNA for 24 h;4,40 and 200 nmol/L of siRNA for 48 h and 72 h(P<0.05).The HCV RNA levels in infected cells treated with IFNα-2b(1 000 IU/ml) for 24 h,48 h and 72 h were significantly lower than those in control groups(P<0.05 or P<0.01).The PKR mRNA levels in Huh-7.5.1 cells transfected with siRNA of three concentrations did not have significant difference,as compared with control groups(P>0.05). Conclusion: siRNA against HCV NS5B region can effectively inhibit HCV replication in HCV infected cells,but can not activate the dsRNA-dependent protein kinase(PKR).更多还原