转化生长因子β3和骨形态发生蛋白2基因共转染骨髓干细胞

Bone marrow stem cells co-transfected with transforming growth factor beta 3 and bone morphogenetic protein 2

背景: 骨形态发生蛋白2及转化生长因子β是骨再生中重要的因子,提高其表达可促进骨髓间充质干细胞的成骨分化。目的: 构建携带转化生长因子β3和骨形态发生蛋白2基因的慢病毒载体,观察其在骨髓间充质干细胞中的表达情况。方法: 应用重组慢病毒技术构建同时携带转化生长因子β3、骨形态发生蛋白2和绿色荧光蛋白基因的重组慢病毒表达载体,并用其转染体外培养的第3代兔骨髓间充质干细胞,以转染携带转化生长因子β3或骨形态发生蛋白2单一基因的慢病毒或单独慢病毒的骨髓间充质干细胞作为对照。转染后1周分别提取各组细胞的总RNA和蛋白进行检测。结果与结论: 荧光显微镜下见转染转化生长因子β3和(或)骨形态发生蛋白2基因3d的骨髓间充质干细胞发绿色荧光,转染效率达90%以上。RT-PCR和Westernblot结果显示,转染转化生长因子β3和骨形态发生蛋白2基因的骨髓间充质干细胞转化生长因子β3和骨形态发生蛋白2mRNA和蛋白的表达均高于单一基因转染组及空白对照组。可见应用慢病毒可成功将转化生长因子β3和骨形态发生蛋白2基因转染至骨髓间充质干细胞并实现其高效表达,且两种基因具有协同促表达作用。

METHODS： The recombinant lentiviral vectors carrying transforming growth factor 63, bone morphogenetic protein 2 and green fluorescent protein were constructed with recombinant lentiviral technology, and then the recombinant lentiviral vectors were used to transfect the passage 3 rabbit bone marrow mesenchymal stem cells in vitro cultured （transfection group）. The bone marrow mesenchymal stem cells transfected with single gene lentivirals （single gone transfection group） carrying transforming growth factor 133 and bone morphogenetic protein 2 or single lentivirals were as control （control group）. At 1 week after trasfection, the total RNA and protein were extracted from each group for detection. RESULTS AND CONCLUSION： The green fluorescence bone marrow mesenchymal stem cells transfected with transforming growth factor 133 and bone morphogenetic protein 2 gene for 3 days could be observed under fluorescence microscope, and the transfection efficiency was over 90%. Reverse transcription-PCR and Western blot results showed the mRNA and protein expressions of transforming growth factor 133 and bone morphogenetic protein 2 in the transfection group were higher than those in the single gene transfection group and the controlgroup. The results indicate that lentivirus can successfully transfect transforming growth factor β3 and bone morphogenetic protein 2 into the bone marrow msenchymal stem cells and achieve its high expression, and these two genes have the synergistic effect of promoting expression.