化学合成siRNA体外对柯萨奇B3病毒的影响

目的RNA干扰是近年来研究基因功能的重要工具。本研究是在培养HELA细胞中，体外化学合成siRNA（small interfering RNA）对柯萨奇B3病毒（Coxsackievirus B3，CVB3）的影响，探讨RNA干扰的抗病毒效应与其应用前景。方法根据siRNA靶序列设计原则，设计了一段针对编码CVB3病毒VP4蛋白基因组的siRNA，并在体外通过化学合成形成siRNA。同时合成一段与CVB基因组序列无关的阴性对照siRNA。用CVB3感染培养HELA细胞，将其分为3组，即siRNA组（n=8），阴性siRNA组（n=8），病毒对照组（n=8），分别观察转染后第1天到第5天的细胞病变，并与未感染CVB3的空白对照组进行比较；采用RT-PCR技术检测感染CVB3各组的病毒RNA片断，观察病毒RNA与内参GAPDH的OD比值；用免疫荧光技术检测各组CVB3蛋白的表达。结果RT-PCR检测各组CVB3病毒5’端非编码区（5’UTR）RNA，siRNA组与阴性siRNA组和病毒对照组比较无明显变化（P〉0．05）；免疫荧光检测各组CVB3病毒蛋白也未见显著差异；细胞病理效应（cytophatic effects，CPE）各组问未见明显差异。结论本文选择针对编码CVB3病毒VP4蛋白基因组的siRNA未能抑制CVB3病毒复制。

Effects of chemically synthetic siRNA on Coxsackievirus B3 in vitro

Objective RNA interference has been an important tool in study of gene function recently.The aim of the study is to explore the effects of chemically synthetic siRNA in vitro on CVB 3 in cultured HELA cells,and to reveal the antiviral effect and the application of RNA interference.Methods On the basis of the principle of target sequence of siRNA,we design a homologous sequence of the genome of CVB 3,which codes viral capsid protein-VP4,and synthesize a siRNA chemically in vitro.We also design a control siRNA that is independent of the genome of CVB 3.We divide HELA cells infected with CVB 3 into 3 groups:siRNA group(n=8),control siRNA group(n=8)and viral control group(n=8).We observe the cytopathic effects(CPE)of these infected groups from the first day to the fifth day respectively,compared with uninfected group(normal control group);utilize RT-PCR to detect the part of RNA of infected groups respectively to observe the OD ratio of viral RNA to GAPDH;and employ immunofluorescence to detect viral protein of each infected group.Results The RNA in the part of 5' untranslated region of CVB 3,measured by RT-PCR,has no statistical significance between siRNA group and control siRNA group or viral control group(p>0.05).The changes of proteins among each infected group,measured by immunofluorescence,have no remarkable difference.The cytopathic effects between siRNA groups and control siRNA group or viral control group have notable difference.Conclusion The study suggests that the siRNA we designed to target homologous genome of CVB 3 which codes viral capsid protein-VP4 does not effectively inhibit the replication of CVB 3.