【摘要】 目的:观察共表达的血管生成素-1(Ang-1)基因和增强绿色荧光蛋白(EGFP)基因的重组慢病毒体外转染大鼠骨髓间充质干细胞(rMSC).方法:体外包装Ang-1基因和EGFP基因共表达的重组慢病毒,测定病毒滴度.转染rMSCs,荧光显微镜下观察EGFP的表达、转导效率,实时荧光定量PCR,Western blot分别在不同时间点行Ang-1基因mRNA以及蛋白表达的相对定量分析.将转染的rMSCs(Ang-1-rMSCs)与对照组rMSCs培养48 h的上清液分别与人脐带静脉内皮细胞(HUVECs)共培养,MTT法检测HUVECs的吸光度值.结果:包装的浓缩慢病毒,滴度为6.1×1010TU/L.转染rMSCs,5 d转染效率为(92.7±3.01)%;实时荧光定量PCR和Western Blot结果显示Ang-1-rMSCs中Ang-1基因mRNA及蛋白表达水平均以转染后3～7 d为高峰,随后开始呈缓慢下降趋势.Ang-1-rMSCs与HUVECs共培养,与对照组rMSCs比较,MTT法检测显示Ang-1-rMSCs能明显促进HUVECs的增殖(P<0.01).结论:在体外重组的慢病毒成功转染rM-SCs,其Ang-1基因表达以转染后3～7 d为高峰,且转染的rMSCs具有Ang-1基因的生物学活性.更多还原

【Abstract】 AIM:To observe coexpressing enhanced green fluorescent protein(EGFP) and rat angiopoietin-1(Ang-1) gene of recombinate lentivirus transfers to the cultured rat bone marrow-derived mesenchymal stem cells(rMSC)in vitro.METHODS: To package the coexpressing EGFP and Ang-1 gene of recombinate lentivirus in vitro.Then the virus titer was examined.The rMSCs were infected by obtained lentiviral particles,the expression of EGFP and the transfection efficiency were examined under fluorescent microscope after transfection.The relative quantitative expression of Ang-1 mRNA and protein were examined at different times by real-time fluorescence quantitative PCR(real-time qPCR)and Western blot.Human umbilical vein endothelial cells(HUVEC) were co-cultured respectively with the supernatant fluid of transfected rMSCs(Ang-1-rMSCs)/rMSCs which had been cultured for 48 h,MTT was used to determine the OD value of HUVECs.RESULTS: The titer of lentiviral vector particles was found to be 6.1×1010 TU/L.The result of the infection in rMSCs showed that the transduction efficiency in Ang-1-rMSCs was(92.7±3.01)% after 5 d;real-time qPCR and Western Blot showed Ang-1 expression had been in the peak during transduction 3-7 d,after then it was in slow downtrend.MTT methods showed the proliferative capacity of HUVECs was better in the co-culture system of Ang-1-rMSCs groups than that of rMSCs groups(P<0.01).CONCLUSION:The recombinate lentivirus can transfer into rMSCs cultured in vitro,in which Ang-1 expression had been in the peak during transduction 3-7 d and the biological effect of Ang-1 gene had been.更多还原