人巨细胞病毒即刻早期基因功能研究  

Immediate-early Gene Function of Human Cytomegalovirus in Virus Replication

作　　者：马晓蒙[1] 顾绍庆[2] 李文静[1] 韦苇[1] 赵媛[1] 

机构地区：[1]江苏大学临床医学院,镇江212001 [2]镇江市第一人民医院儿科,212001

出　　处：《医学研究杂志》2014年第9期44-48,共5页Journal of Medical Research

基　　金：江苏省自然科学基金资助项目(BK 2008238)

摘　　要：目的探讨人巨细胞病毒(human cytomegalovirus,HCMV)在体外增殖过程中即刻早期(immediately early,IE)基因的作用。方法设计并化学合成3条靶向IE基因外显子序列的siRNA,应用阳离子脂质体法将干扰序列转染入人胚肺成纤维(human embryonic lung fibroblast,HELF)细胞,用HCMV感染已转染入siRNA的细胞,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。采用流式细胞术检测转染效果,荧光定量PCR和琼脂糖凝胶电泳方法检测分析导入的siRNA对IE基因和GAPDH阳性对照基因的抑制效果以及早期(E)基因和晚期(L)基因的表达情况。结果 siRNA成功导入HELF细胞内,6孔板内5μl脂质体可导入的最佳siRNA量约为100pmol,阳性对照组GAPDH表达量较其他对照组有明显的下降(P<0.05),抑制率为70.8%;干扰组中3条siRNA对IE基因的表达均有抑制作用。IE基因表达下调后,E基因和L基因的表达较对照组也有明显下降(P<0.05)。结论在体外,靶向IE基因的siRNA能有效抑制IE基因的表达从而影响E基因和L基因的表达,表明IE基因的有效表达是E基因和L基因表达的基础。Objective To investigate the effect of human cytomegalovirus immediate early gene（IE） on the replication of virus using RNA interference.Methods Three small interfering RNAs （siRNAs） were designed and synthetized according to the sequence of HCMV -IE.The siRNAs were transfected into HELF cells by cationic liposome.Then cells with siRNA were infected by HCMV.At the same time,the normal control group,negative control group,positive control group and transfection reagent group in experiment were set.The effects of transfection were detected by flow cytometry.And the inhibitive effect of siRNAs were examined by agarose gel electrophoresis and fluorescent quantitation PCR.Results siRNAs were successfully transfected into HELF cells,and the best concentration of siRNAs was 100pmol when we used 5μl the cationic liposome to transfect.Expression of GAPDH in positive control group had a dramatic decline compared with other control group.Three siRNAs all had inhibitive effect,and siRNA-2 was the best one whose inhibition ratio was 70.8％.With the reduced expression of IE,the expression of E gene and L gene had obviously decline （P ＜ 0.05）.Conclusion siRNAs targeting IE could inhibit the expression of IE effectively,which resulted in the decline of E gene and L gene.As a result,the viral load would be drop.From the above,the expression of E gene and L gene was based on the effective expression of IE gene.

关 键 词：人巨细胞病毒 即刻早期基因 干扰RNA 基因表达 

分 类 号：R3[医药卫生—基础医学]