bFGF基因体外转染兔骨髓间充质干细胞及对其增殖的影响

Construction of Bone Mesenchymal Stem Cells(bMSCs) Transfected with bFGF in Vitro and the Enhancement of bMSCs

目的:观察bFGF基因体外转染兔骨髓间充质干细胞(bMSCs)后细胞目的基因的表达及对其增殖的影响。方法:将兔骨髓来源的间充质干细胞分离培养扩增,用PCR扩增法获得绿色荧光蛋白-bFGF基因重组质粒,转染间充质干细胞,用荧光显微镜观察转染后绿色荧光蛋白基因的表达,计算转染率,用MTT检测转染细胞的增殖特性。结果:实验成功地构建了绿色荧光蛋白-bFGF基因重组质粒,倒置荧光显微镜下观察到转染后的bMSCs发出稳定的绿色荧光信号,转染率为(43.32±4.51)%,MTT法显示重组质粒组增殖速度显著高于空白质粒组和未转染细胞组(P<0.05)。结论:运用转染的方法实现了两种基因在MSCs中的共同表达,为监控MSCs细胞的转染和表达过程提供了一种手段。为后期利用MSCs细胞进行组织工程骨组织制造和移植提供了规范化的监控和程序化生产手段。

Objective： To construct a retroviral vector carrying both enhanced green fluorescent protein（EGFP） and human basic fibroblast growth factor（bFGF） gene for observing the enhanced effect of transfected bone mesenchymal stem cells（bMSCs） and gene expression.Methods： bMSCs were constructed from bones of rabbits.The pIRES2-EGFP-bFGF-pcDNA3 expression plasmid was transfected into bMSCs with liposome lipofectamine.The expression of EGFP was observed by inverted fluorescence microscope and was calculated by transfection efficiency.The proliferativity of the transfected cells was detemined with MTT assay.Results： The recombinant retroviral vector bFGF-pcDNA3 and pIRES2-EGFP was successfully constructed.The bMSCs infected by both vectors stably gave out green fluorescence under microscope.The transfection efficiency was（43.32±4.51）%.Transfected cells grew more quiclkly than that of non-transfected cells（P0.05）.Conclusion： Rabbit bMSCs can be successfully transfected by using pIRES2-EGFP-bFGF-pcDNA3 plasmid.This vector can be easily traced.