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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。
Objective: To construct a lentiviral vector containing the mouse insulin-like growth factor-1 (IGF-1) gene and identify its expression in neural stem cells (NSCs). Methods: IGF-1 gene was amplified from mouse skeletal muscle cells by RT-PCR and inserted into pcDNA3.1 plasmid to construct lentiviral vector pXZ9-IGF-1. 293FT cells were transfected with liposome-mediated three-plasmid co-transfection to obtain the virus supernatant. Neural stem cells were isolated and cultured, and immunofluorescence staining was used to identify Nestin. The lentivirus infected neural stem cells. The expression of GFP was observed under the microscope. The expression of IGF-1 was detected by RT-PCR and ELISA kit respectively from the mRNA and protein levels. Results: IGF-1 gene was amplified from mouse skeletal muscle cells by RT-PCR and cloned into pcDNA3.1 vector. The lentiviral plasmid pXZ9-IGF-1 was successfully constructed by subcloning. 293FT cells were transfected with plasmid by high-titer Lentiviral Lentiviral Particles, in vitro infection of neural stem cells efficiently. After infection of neural stem cells, IGF-1 mRNA and protein in the culture medium were both highly expressed. Conclusion: The lentiviral vector pXZ9-IGF-1 containing mouse IGF-1 gene was successfully constructed and expressed in neural stem cells.