稳定转染增强型绿色荧光蛋白大鼠骨髓间充质干细胞系的建立

背景：利用间充质干细胞或含有治疗因子的干细胞进行有选择性的杀伤肿瘤细胞是一种有前途的治疗方法。目的：建立含稳定转染增强型绿色荧光蛋白的大鼠骨髓间充质干细胞系。方法：通过脂质体介导慢病毒质粒pVector-EGFP、pHelper、Envelope共转染293T细胞完成载体病毒构建,以实时荧光定量PCR检测慢病毒滴度;取对数生长期的SD大鼠骨髓间充质干细胞,以复感染指数MOI值0,5,10,15,20加入携带报告基因增强型绿色荧光蛋白的慢病毒载体稀释液,72h后观察各组增强型绿色荧光蛋白的表达效率及阳性转染率。结果与结论：携带增强型绿色荧光蛋白的慢病毒载体系统转染293T细胞能够正确表达,滴度为1×108TU/mL。包装好的病毒颗粒转染大鼠骨髓间充质干细胞二三天后,各孔均有增强型绿色荧光蛋白的表达。MOI值从0增至10,细胞的阳性表达率逐渐提高（P〈0.05）,MOI值为10的组能获得〉70%的转染率,但MOI值从10增至20,转染率变化不明显。说明以MOI值为10的滴度将慢病毒载体可将外源基因高效转入大鼠骨髓间充质干细胞内,建立含稳定转染增强型绿色荧光蛋白的大鼠骨髓间充质干细胞系。

Establishment of rat bone marrow mesenchymal stem cell lines stably transfecting enhanced green fluorescent protein

BACKGROUND：The use of mesenchymal stem cells or therapeutic factor-containing stem cells is a promising therapeutic method to selectively kill tumor cells.OBJECTIVE：To establish rat bone marrow mesenchymal stem cell lines stably transfecting enhanced green fluorescent protein.METHODS：The lentiviral plasmid pVector-EGFP with the packaging plasmid pHelper 1 and the envelop plasmid pHelper 2 were co-transfected into 293T cells.The lentiviral titer was detected by real-time,fluorescence-based quantitative PCR.SD rat bone marrow mesenchymal stem cells during logarithmic phase were seeded in 24-well plates and exposed to lentiviral vector coding for EGFP reporter gene at multiplicity of infection（MOI） of 0,5,10,15 and 20,respectively.EGFP expression and transfection efficiency were determined 72 hours later.RESULTS AND CONCLUSION：The lentiviral vector system carrying EGFP was successfully tranfected into 293T cells with a lentivirual titer of 1×108 TU/mL.At 2-3 days after rat bone marrow mesenchymal stem cells were transfected with packaged lentiviral vector coding for EGFP reporter gene,EGFP expression was detected in each well.MOI values ranged from 0 to 10.EGFP-expressing cells were gradually increased（P 0.05）.Over 70% cells were EGFP-positive at 3 days after transfection by lentiviral vector at MOI of 10.A significant dose-response was observed with increasing lentiviral titer for MOI 0 to 10（P 0.05）,whereas the ratio of transfection decreased with increasing lentiviral liter for MOI 10 to 20.These findings suggest that lentivirus is an ideal vector for gene transduction and it can transduce an exogenous gene into rat bone marrow mesenchymal stem cells with over 70% efficency at the MOI of 10 to establish rat bone marrow mesenchymal stem cell lines stably transfecting EGFP.