人脐静脉内皮细胞Caveolin-1基因沉默模型的建立

目的:利用小干扰RNA(siRNA)技术建立人脐静脉内皮细胞(ECV304)小凹蛋白(Caveolin-1)基因沉默模型,为研究Caveolin-1基因在人脐静脉内皮细胞中的作用.方法:通过Ambion专用软件对Caveolin-1基因编码区分析,设计合成短发夹RNA(shRNA)单链.通过T4连接酶将退火反应合成的shRNA双链克隆入含RNA PollII聚合酶表达元件的pENTRTM/U6入门载体,并利用Gateway技术构建相应的慢病毒RNA干扰(RNAi)表达载体.用REAL-TIME RT-PCR、蛋白印迹法分析沉默效率.结果:测序结果显示pENTRTM/U6载体插入的Cavelin-1基因shRNA序列大小及阅读框架正确.LR重组反应后成功构建了针对Caveolin-1基因的RNAi慢病毒表达系统.转染ECV-304细胞获得对Caveolin-1的沉默效率大于85%.结论:成功地构建了针对Caveolin-1基因的RNAi慢病毒表达系统,获得了长期稳定的基因剔除效应.人脐静脉内皮细胞Caveolin-1基因沉默模型能长期保种及传代,为进一步研究Cav-eolin-1基因的功能奠定了基础.

Constructing Caveolin-1 silencing model in human umbilical vein vascular endothelial cell

Objective：To build the Caveolin-1 silencing model in human umbilical vein vascular endothelial cell（ECV304） using the siRNA silencing technology to make the foundation for studying the Caveolin-1′s function in the human umbilical vein vascular endothelial cell.Methods：The coding region of Caveolin-1 was analysed to design short hairpin RNA（shRNA） single strand by the professional software Ambion.After annealing to form shRNA double strand,it was cloned into the pENTR？/U6 entry vector containing RNA polymerase III express element by T4 ligase and then generate the lentiviral expressing vector.Silencing efficiency was identified by real-time RT-PCR and Western blot.Results：The sequencing results showed the pENTRTM/U6 gate vector including the shRNA of Cavelin-1 was correct.We constructed the Caveolin-1 RNAi lentiviral expression system successfully after the LR recombination reaction.The expression of Caveolin-1 in the siRNA-transfected ECV304 cells were silenced to the extent of more than 85%.Conclusion：We construct the Caveolin-1 RNAi lentiviral expression system successfully,and get a long-term and stable gene silencing effect.The Caveolin-1 silencing model in human umbilical vein vascular endothelial cell can keep and passage for a long time,which makes foundation for further studying the Caveolin-1′ s function.