联合G418筛选构建GFP转基因骨髓基质干细胞系的实验研究
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摘要
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.
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.
引文
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[10]何玉祥,孙岩,刘振川,等.大鼠骨髓基质干细胞体外分离、纯化、培养的实验研究[J].解放军医药杂志, 2014, 26(5):10-13.
[11]李嘉琪,李紫聪,吴珍芳,等. G418处理核供体细胞对猪克隆胚胎体外发育效率的影响[J].华南农业大学学报, 2016, 37(5):13-18.
[2] Jiang Y, Jahagirdar BN, Reinhardt RL, et al. Pluripotency of mesenchymal stem cells derived from adult marrow[J]. Nature, 2002, 418(6893):41-49.
[3]管利娜,陈燕红,朱恒涛,等.小鼠诱导性多潜能干细胞向内耳毛细胞和螺旋神经节细胞分化的体外研究[J].中华耳鼻咽喉头颈外科杂志, 2014, 49(8):680-686.
[4] Varghese HJ, Davidson MT, MacDonald IC, et al. Activated ras regulates the proliferation apoptosis balance and early survival of developingmicrome tastases[J]. Cancer Res, 2002, 62(3):887-891.
[5] Swanson CI, Hinrichs T, Johnson LA, et al. A directional recombinationcloning system for restriction-and ligation-free construction of GFP, DsRed, and lacZ transgenic Drosophila reporters[J]. Gene,2008, 408(1-2):180-186.
[6] Varghese HJ, Davidson MT, MacDonald IC, et al. Activated ras regulates the proliferation apoptosis balance and early survival of developingmicrome tastases[J]. Cancer Res, 2002, 62(3):887-891.
[7]吴伶俐,孙永强,王建红,等.小鼠黑色素瘤B16F10-Red细胞株的建立与生物学特性分析[J].现代肿瘤医学, 2016, 24(6):861-864.
[8]李艳,陆伟,竺丽梅,等.慢病毒介导的GFP转染对小鼠骨髓间充质干细胞表型、增殖和分化能力的影响[J].南京大学学报(自然科学), 2014, 50(6):883-889.
[9]王亭忠,马爱群,蒋文慧,等.大鼠骨髓间充质干细胞的分离培养及GFP转染标记[J].西安交通大学学报(医学版), 2005, 26(5):431-434.
[10]何玉祥,孙岩,刘振川,等.大鼠骨髓基质干细胞体外分离、纯化、培养的实验研究[J].解放军医药杂志, 2014, 26(5):10-13.
[11]李嘉琪,李紫聪,吴珍芳,等. G418处理核供体细胞对猪克隆胚胎体外发育效率的影响[J].华南农业大学学报, 2016, 37(5):13-18.
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