RNA干扰技术在体外抑制人巨细胞病毒基因表达的应用

In vitro inhibition of the human cytomegalovirus gene replication by RNA interference

目的探讨RNA干扰技术在体外抑制人巨细胞病毒(HCMV)基因表达时siRNA转染和病毒感染的先后顺序对抑制效果的影响。方法设计并化学合成靶向即刻早期(IE)基因的siRNA。应用阳离子脂质体法将siRNA导入人胚肺成纤维(HELF)细胞,并分为先转染siRNA后感染HCMV、先感染HCMV后转染siRNA以及同时转染siRNA和感染HCMV3组,同时设置正常对照组、阴性对照组、阳性对照组和转染试剂对照组。采用荧光定量PCR和琼脂糖凝胶电泳方法分别检测分析各组转染的siRNA对IE基因和阳性对照基因GAPDH的抑制效果。结果阳性对照组GAPDH基因mRNA表达量较阴性对照组和转染试剂对照组有明显下降,siRNA抑制率为70.4%,差异有统计学意义(P<0.05)。先转染si RNA后感染HCMV组、先感染HCMV后转染si RNA组、同时转染siRNA和感染HCMV组以及阳性、阴性、转染试剂对照组的IE基因m RNA表达量比较,差异有统计学意义(F=146.93,P=0.000)。其中先转染siRNA后感染HCMV组以及同时转染si RNA和感染HCMV组对IE基因m RNA表达的抑制效果均好于先感染HCMV后转染siRNA组,差异有统计学意义(P<0.05)。结论在体外,siRNA能有效抑制靶基因的表达,且先转染si RNA再感染病毒的抑制效果相对更好,提示siRNA对HCMV感染具有一定的预防作用。

Objective To investigate the effect on in vitro inhibition of the human cytomegalovirus （HCMV） gene replication by RNA interference using different orders of small interfering RNA （siRNA） transfection and HCMV infection. Methods The siRNAs were designed and synthesized according to the sequence of the immediate early （IE） genes of HCMV. The siRNAs were transfected into human embryonic lung fibroblastic （HELF） cells by cationic liposome through different orders including before or after or at the same time as HCMV infection. Simultaneously, the control groups were set including normal control group, negative control group, positive control group and transfection reagent group. The inhibitory effects of siRNAs on HCMV-IE and a positive control gene of GAPDH were examined by lfuorescent quantitative PCR and agarose gel electrophoresis. Results The expression level of GAPDH mRNA in positive control group was signiifcantly lower than that in negative control group and transfection reagent group （P〈0.05）, and the inhibition rate of siRNA was 70.4%. Among different experimental groups of siRNAs transfection before, after or at the same time as HCMV infection and three control groups of negative control group, positive control group and transfection reagent group, the expression level of IE mRNA was signiifcantly different （F=146.93, P=0.000）. In the groups of siRNAs transfection before HCMV infection or at the same time as HCMV infection, the expression levels of IE mRNA were signiifcantly lower than those in the group of siRNAs transfection after HCMV infection （P〈0.05）. Conclusions In vitro, siRNAs can effectively inhibit the expression of target gene and it is better to do siRNA transfection ifrst then followed by HCMV infection. It is suggested that siRNA might have a role in prevention from HCMV infection.