化学合成siRNA体外对柯萨奇B_3病毒的影响  

Effects of chemically synthetic siRNA on Coxsackievirus B_3 in vitro

作　　者：丁英俊[1] 刘松兰[1] 廖玉华[1] 董继华[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院心研所心血管免疫实验室,武汉市430022

出　　处：《中国分子心脏病学杂志》2004年第4期204-208,共5页Molecular Cardiology of China

摘　　要：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 (smallinterferingRNA)对柯萨奇B3病毒 (CoxsackievirusB3,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 0 5 ) ;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 (cytophaticeffects,CPE)各组间未见明显差异。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。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 CVB3 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 CVB3, which codes viral capsid pmtein-VP4, and synthesize a siRNA chemically in vitro. We also design a control siRNA that is independent of the genome of CVB3. We divide HELA cells infected with CVB3 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 CVB3, 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 CVB3 which codes viral capsid protein-VP4 does not effectively inhibit the replication of CVB3.

关 键 词：柯萨奇病毒B3(CVB3) RNA干扰 SIRNA 

分 类 号：R346[医药卫生—基础医学]