摘要
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 recombinant heme oxygenase-1 （HO-1） gene-transfected porcine mesenchymal stem cells（MSCs） in vetro in gene tharapy. Methods Lentiviral system was utilized to introduce HO-1 gene into MSCs isolated from porcine bone marrow and cultured in vitro. RT-PCR and GFP marker were used to determine the expression of HO-1. MTT and trypan blue staining were used to detect the proliferative capacity of the MSCs. Results MSCs were infected with lcntivirus at a multiplicity of infection（MO I） of 20 with optimal expression efficiency of 80%. The expressions of CD44 and CD105 on surface of MSCs were observed at high levels. GFP marker was observed at 96 hours after gene transfection and then gradually enhanced. The expression of HO-1 mRNA appeared in the transfected cells. HO-1 transfection did not show any significantly inhibitory effect on MSCs. Conclusion The expression of HO-1 is up-regulated in MSCs transfected successfully by lentiviral vector and the transfection has no significant effects on the survival and proliferation of MSCs.
出处
《江苏医药》
CAS
CSCD
北大核心
2012年第15期1750-1753,F0002,共5页
Jiangsu Medical Journal
基金
苏州市科教兴卫基金(SWKQ0812)
