摘要
本研究探讨多药耐药(mdr1)基因体外转染人骨髓间充质干细胞(BMMSC)以应用于基因治疗的可行性、安全性。体外分离、培养、鉴定MSC;采用具有高效转染非分裂期细胞的慢病毒载体(lentiviral vector,LV)系统将mdr1基因导入BMMSC中;采用RT-PCR和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。结果表明:慢病毒感染MSC的感染复数(multiplicity of infection,MOI)为10,最佳感染率可达80%;MSC表面低表达CD34、HLA-DR、CD31、CD45,高表达CD44、CD105、CD90、CD13;GFP荧光表达自72小时开始出现,以后逐渐增强;转染细胞中显示目的基因mRNA的表达;转染对MSC存活及增殖几乎无影响(p>0.05)。结论:慢病毒载体可成功转染人骨髓间充质干细胞并使其mdr1表达增高,转染对MSC存活及增殖基本无影响。
This study was aimed to investigate the feasibility and security of mdrl gene-modified mesenchymal stem cells （MSCs） so as to establish the experimental foundation for gene therapy. Lentiviral system was utilized to introduce the mdrl gene into MSCs which were isolated from human bone marrow and cultured in vitro; RT-PCR and GFP marker were used to determine the expression of mdrl ; MTT and trypan blue staining were used to detect the proliferative capacity of the MSCs. The results indicated that MSCs were infected with lentivirus at a multiplicity of infection （MOI） of 10 with optimal expression efficiency of 80% ; the expressions of CD34, HLA-DR, CD31 and CD45 on surface of MSCs were found at low levels, however, the expressions of CD44, CD105, CD90 and CD13 on surface of MSCs were observed at high levels; GFP marker was observed on 72 hours after gene transfection and then gradually was enhanced; the expression of mdrl mRNA appeared in transfected cells; Mdrl transfection did not show a significantly inhibitory effect on MSCs. It is concluded that the expression of mdrl is up-regulated in MSCs transfected cuccessfully by lentiviral vector, and the transfection has no significantly effects on survival and proliferation of MSCs.
出处
《中国实验血液学杂志》
CAS
CSCD
2009年第3期690-694,共5页
Journal of Experimental Hematology
