慢病毒介导RNA干扰沉默Fas基因在脐带间充质干细胞中的表达

Lentivirus-mediated stable Fas gene silencing in human umbilical cord-derived mesenchymal stem cells

目的应用慢病毒介导的RNA干扰技术,构建Fas基因的siRNA慢病毒表达载体,建立Fas基因稳定干扰的人脐带间充质干细胞(UC-MSCs)株,为下一步使用低表达Fas基因的UC-MSCs治疗再生障碍性贫血奠定实验基础。方法以人Fas基因mRNA序列作为干扰靶点,设计4组靶向Fas的shRNA序列,合成寡核苷酸,与经过BamHI、EcoRI双酶切后的LV3载体连接获得重组慢病毒,通过感染293T细胞对慢病毒进行包装和滴度测定。体外培养人UC-MSCs,使用包装好的慢病毒感染UCMSCs,应用Real time-PCR和Western blotting检测感染后细胞中Fas mRNA及蛋白的表达情况。结果经酶切以及基因测序鉴定证明慢病毒载体构建成功,病毒悬液滴度为3×108TU/ml。Real time-PCR和Western blotting结果显示干扰组细胞的Fas表达水平较对照组显著降低。结论慢病毒介导的SiRNA能有效沉默UC-MSCs中Fas基因的表达。

Objective To construct a lentivirus- mediated vector for RNA interference（RNAi） of Fas and establish a human umbilical cord-derived mesenchymal stem cells（UC-MSCs） line with stable Fas gene silencing. Methods Four short hairpin RNA sequences targeting the coding region of human Fas mRNA were designed. The synthesized oligonucleotides were ligated with the lentivirus vectors harvested from BamHI and EcoRI double digestion of LV3 recombinant vector. The recombinant lentivirus vectors were transfected into the packaging cells 293 T, and the lentivirus titers were determined.Cultured UC- MSCs were infected with the lentivirus, and real- time PCR and Western blotting were used to detect the expressions of Fas mRNA and protein in the transfected cells. Results Restriction digestion and DNA sequencing showed that the lentiviral vectors were successfully constructed, and the titer of lentivirus reached 3×108TU/ml in the packaging cells. Realtime PCR and Western blot demonstrated significantly suppressed Fas gene expression in UC-MSCs after infection with the recombinant lentivirus. Conclusion Lentivirus- mediated RNAi can effectively inhibit Fas gene expression in cultured UCMSCs.