腺病毒介导血管内皮细胞生长因子基因体外转染人脐带间充质干细胞

目的探讨腺病毒介导血管内皮细胞生长因子(VEGF)基因转染人脐带间充质干细胞(UCMSCs)后目的基因表达情况及对UCMSCs生长增生的影响。方法 UCMSCs传代培养后,以腺病毒介导绿色荧光蛋白(GFP)基因进行体外转染,倒置荧光显微镜下观察细胞转染效果,流式细胞仪检测细胞转染效率,确定最佳的病毒感染复数(MOI)。将实验分为VEGF基因转染组和未转染组。转染组在最佳MOI值条件下以腺病毒介导VEGF基因体外转染UCMSCs;未转染组以PBS代替病毒液。通过免疫荧光染色及Western blotting检测两组UCMSCs VEGF蛋白的表达情况,RT-PCR检测VEGF基因mRNA的表达情况,ELISA检测培养液中VEGF浓度,MTT法评价转染后对UCMSCs生长增生的影响。结果腺病毒介导的GFP基因对于UCMSCs具有较高的转染效率,转染效率与MOI值具有量效关系,当MOI为100倍时,转染效率达95%。转染VEGF基因后2d,UCMSCs在mRNA和蛋白水平上均可有效表达VEGF,免疫荧光染色、RT-PCR、Western blotting显示转染组明显表达VEGF,ELISA检测结果显示7d时达到表达高峰,13d后仍可检测到VEGF的表达。介导VEGF基因转染的腺病毒对UCMSCs的生长增生没有明显影响。结论 UCMSCs是一种较理想的基因载体细胞,腺病毒介导VEGF基因可以有效转染UCMSCs,转染后VEGF基因可获得较高的表达水平。

Transfection of adenovirus containing vascular endothelial growth factor gene into umbilical cord mesenchymal stem cell in vitro

Objective To investigate the expression of adenovirus transfected vascular endothelial growth factor（VEGF）in umbilical cord mesenchymal stem cells（UCMSCs）in vitro and the effect of transfection on UCMSCs proliferation.Methods Adenoviral vector containing hVEGF165 gene was constructed and UCMSCs were passaged and expanded.The infection efficiency of adenovirus vector to UCMSCs was tested by Ad.GFP infection procedure.GFP expression efficiency was observed using the fluorescence microscope and flow cytometry.The experiment was divided into two groups, namely,the VEGF transfection group and non-transfection group.Ad.VEGF was transfected into the UCMSCs by adenovirus vector.The nontransfected UCMSCs were used as the controls.VEGF expression in UCMSCs and its secretion in culture medium were measured by immunohistochemical staining, RT-PCR,Western blotting and ELISA,respectively.Influence of recombinant adevirous on cell activity was evaluated by MTT.Results UCMSCs could be effectively infected by adenovirus containing GFP gene in vitro,thetransfection efficiency had the dose-effect relationship with multiplicities of infection（MOI）. When MOIwas 100,the infection efficiency was morethan 95%.The expression of VEGF was traced both in cell lysate and in culture medium.A maximum productionofVEGF was observed at 5-9days after infection,peakingonday 7,and VEGF was found evenon day 13.There was no effect on the growth and proliferation of UCMSCs transfected by recombinant adevirus.Conclusions Gene transfer technology mediated by adenoviral vector can transfect VEGF gene into UCMSCs with high efficiency.UCMSCs transfected by VEGF gene could express higher VEGF during two weeks.