人bFGF重组慢病毒转染BMSCs的实验研究

Experimental Study on bFGF Gene Lentiviral Vector Transfecting BMSCs

目的探讨携带人bFGF和GFP基因慢病毒(lenti-bFGF-GFP)转染骨髓间充质干细胞(BMSCs)及不同MO(Imultiplicity of infection)的转染效率。方法实验组:带有bFGF和GFP基因慢病毒感染的BMSCs(bFGF-GFP-BMSCs)。空载体组:带GFP基因慢病毒感染的BMSCs(GFP-BMSCs)。空白组:未经感染的BMSCs;ELISA、RT-PCR检测各组细胞培养72h后bFGF的表达。MOI分别为10、30、50转染BMSCs的转染率。结果bFGF-GFP-BMSCs组表达bFGF蛋白量经ELISA、RT-PCR检测均明显高于GFP-BMSCs组和BMSCs组。MOI为10、30、50、的转染效率分别为36.6%、84.2%、98.5%。结论带有bFGF和GFP基因慢病毒成功转染骨髓间质干细胞并表达,为bFGF基因修饰的骨髓间充质干细胞移植治疗脊髓损伤奠定了基础。MOI为50时,BMSCs的转染率超过85.0%。

Objective To explore the ways of bFGF and green fluorescent protein（GFP） gene highly transfected to MSCs by lenti-bFGF -GFP and the efficiency of different MOI（muhiplicity of infection ）transfected to MSCs. Methods MSCs was subject to different treatments with three groups,that was bFGF-GFP-BMSCs group, GFP-BMSCs group and BMSCs group. 72 hours after culture of MSCs,ELISA and RT-PCR was adopted to quantify the amount of protein and mRNA of bFGF in the different groups. The transfection of different MOI（10,30,50） was transfected into BMSC. Results Both ELISA and RT-PCR experiments discovered this：72 hours after transfection, the amount of bFGF protein of bFGF-GFP-BMSCs group was much higher than these of the GFP-BMSCs group and BMSCs group. When the MOI equal to lO,30,50,the efficiency of lentivirus transfected into MSCs was 36.6% ,84.2% ,98.5%. Conclusion Lentivirus mediated by bFGF gene and GFP gene can infect MSCs successfully and which can express and secret bFGF protein after transfection. MSCs infected by lentivirus which was mediated by bFGF gene and GFP gene is an efficient way for gene therapy of spinal cord injury（SCI）. When MOI equal to 50,the efficiency exceeded 85.0%.