携带小鼠IGF-1基因的慢病毒载体构建及其在神经干细胞中的表达

目的:构建含有小鼠胰岛素样生长因子-1(IGF-1)基因的慢病毒载体,鉴定其在神经干细胞(NSCs)中的表达。方法:采用RT-PCR方法从小鼠的骨骼肌细胞中扩增IGF-1基因,克隆入pcDNA3.1质粒,构建慢病毒载体pXZ9-IGF-1。用脂质体介导三质粒共转染法转染293FT细胞,获得病毒上清。分离培养神经干细胞,Nestin免疫荧光化学鉴定。慢病毒感染神经干细胞,显微镜下观察GFP表达情况,通过RT-PCR及ELISA试剂盒分别从mRNA和蛋白水平检测IGF-1的表达。结果:通过RT-PCR法从小鼠的骨骼肌细胞中扩增出IGF-1基因并克隆入pcDNA3.1载体,经亚克隆成功构建慢病毒质粒pXZ9-IGF-1。质粒经脂质体转染293FT细胞获高滴度慢病毒颗粒,体外高效感染神经干细胞。神经干细胞经感染后,IGF-1基因mRNA和培养基中蛋白水平均高表达。结论:成功构建含小鼠IGF-1基因的慢病毒载体pXZ9-IGF-1,并在神经干细胞内获得表达。

Construction of lentiviral vector carrying mouse IGF-1 gene and expression of IGF-1 in neural stem cells

Objective: To construct a lentiviral vector carrying mouse insulin-like growth factors(IGF-1) gene,and to detect the expression of IGF-1 in the neural stem cells(NSCs).Methods: IGF-1 fragment was amplified from skeletal muscle of mouse by RT-PCR and cloned into pcDNA3.1 vector and then constructed into the lentiviral vector pXZ9-IGF-1.The recombinant lentivirus were produced by co-transfected three plasmids into 293FT cells using lipofectamine 2000.To isolate and culture NSCs which were detect with Nestin immunofluorescent staining.The NSCs were infected by the concentrated lentivirus,GFP expression was examined under a fluorescent microscope and the expression of IGF-1 mRNA was detected by RT-PCR and the level of protein was detected by ELISA.Results: The IGF-1 fragment was amplified from skeletal muscle of mouse by RT-PCR and then constructed into pcDNA3.1 vector and recombinant lentiviral vector pXZ9-IGF-1 was constructed successfully.High titer lentivirus were attained from 293FT cells after the plasmids were transfected and which infected the NSCs efficiently in vitro.After the NSCs being infected,the level of mRNA and the protein in culture medium of IGF-1 gene were high expression.Conclusion: The lentiviral vector pXZ9-IGF-1 containing mouse IGF-1 gene is constructed successfully,and IGF-1 gene can express efficiently in NSCs.