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目的探讨重组血红素氧合酶1(HO-1)基因体外转染猪骨髓间充质干细胞(MSCs)应用于基因治疗的可行性。方法体外分离、培养并鉴定MSCs。采用具有高效转染非分裂期细胞的慢病毒载体系统将HO-1基因导入MSCs中;采用RT-PCR和绿色荧光蛋白(GFP)荧光技术检测目的基因的表达,台盼蓝染色及MTT法检测转染后细胞的增殖能力。结果慢病毒感染MSCs的感染复数为20,最佳感染率可达80%;MSCs表面高表达CD44和CD105;GFP荧光表达自96h开始逐渐增强;转染细胞中显示目的基因mRNA的表达;转染对MSCs存活及增殖几乎无影响。结论慢病毒载体可成功转染猪MSCs,并使其HO-1表达增高,转染对MSCs存活及增殖基本无影响。
Objective To investigate the feasibility of transfection of recombinant heme oxygenase-1 (HO-1) gene into pigs for gene therapy in vitro. METHODS: MSCs were isolated, cultured and identified in vitro. HO-1 gene was transfected into MSCs by lentiviral vector system with efficient transfection of non-dividing cells. The expression of HO-1 gene was detected by RT-PCR and green fluorescence protein (GFP) fluorescence assay, trypan blue staining and MTT assay The ability of cells to proliferate after transfection. Results The number of infected lentivirus-infected MSCs was 20 and the optimal infection rate was 80%. The expression of CD44 and CD105 on the surface of MSCs was enhanced. The expression of GFP began to increase from 96h. The transfected cells showed the expression of target gene mRNA. There is almost no effect on MSCs survival and proliferation. Conclusion The lentiviral vector can successfully transfect porcine MSCs and increase the expression of HO-1. Transfection has no effect on the survival and proliferation of MSCs.