联合G418筛选构建GFP转基因骨髓基质干细胞系的实验研究

Experimental study on constructing bone marrow stromal stem cells line in transgenic SD rats expressing green fluorescent protein by G418 screening

目的联合G418筛选构建绿色荧光蛋白(GFP)转基因SD大鼠骨髓基质干细胞(BMSCs)系,用于骨髓基质干细胞体外培养及向内耳神经细胞诱导分化的进一步研究。方法制备大鼠骨髓基质干细胞,选用脂质体Lipofectamine^(TM)2000转染方法将含有pcDNA3.1-GFP的重组质粒转染至大鼠骨髓基质干细胞中,荧光显微镜检测GFP蛋白表达,通过G418筛选法建立稳定、含pcDNA3.1-GFP的单克隆骨髓基质干细胞系。结果 500μg/mL的G418压力筛选后,获得了具有G418抗性的绿色荧光蛋白(GFP)转基因SD大鼠骨髓基质干细胞系。结论成功地培育了G418抗性的GFP转基因大鼠骨髓基质干细胞系,为进行下一步体外特异微环境下的诱导分化提供实验基础。

Objective To construct bone marrow stromal stem cells（BMSCs） in transgenic SD rats expressing green fluorescent protein（GFP） by G418 screening, in order to provide basis for in vitro culture of BMSCs and induced differentiation of BMSCs into inner ear nerve cells. Methods Recombinant plasmid containing pcDNA3.1-GFP was transfected into the rats, BMSCs prepared by Lipofectamine-（TM）2000 transfection, and the expression of GFP protein was detected by fluorescence microscopy. A line of stable and monoclonal bone BMSCs containing pcDNA3.1-GFP was established by G418 screening. Results After pressure screening with 500 μg/mL G418,BMSCs with G418 resistance in transgenic SD rats expressing GFP were obtained. Conclusion The lines of BMSCs of transgenic SD rats expressing GFP with G418 resistance were successfully cultured, which provides a basis for the induced differentiation in vitro.