【作者】 潘求真； 孙书锋； 韩红兵； 张宝路； 李海； 田亮； 李佳； 徐曙光； 连正兴； 杨宁；

【Author】 Pan Qiuzhen, Sun Shufeng, Han Hongbing, Thong Baolu, Li Hai, Tian Liang, Li Jia, Xu Shuguang, Lion Zhengxing, Yang Ning (China Agricultural University, Beijing, 100094 Northeast Agricultural University, Harbin, 150030)

【机构】 中国农业大学动物科技学院；

【摘要】 目的采用电穿孔法将带有GFP的质粒(pGFP-NEO)转染MSCs,经过G418筛选阳性克隆,用荧光显微镜检测GFP的表达情况。结果以电压150V,20ms一次脉冲进行转染。转染 GFP质粒的MSCs能在含有6418的培养基中生长,而未转染的MSCs不断死亡。转染24h后即能观察到少量荧光细胞,一个月后大部分荧光细胞呈集落样生长,荧光表达较强,转染率为25％- 30％。稳定表达的阳性克隆细胞,已稳定表达6个月。结论 GFP基因能在MSCs中进行有效的可持续、稳定、高效表达;因此,转染GFP的MSCs能作为研究MSCs的诱导分化及生物细胞工程治疗的载体细胞。更多还原

【Abstract】 Objective The transfected pig MSCs were obtained with pGFP-NEO vector containing the GFP gene and Neo gene by electroporation. The MSCs expressing GFP were detected by fluorescent microscopy and through G418 selecting and cloned culture in limiting dilution. Results In the optimum electroporation condition: voltage at 150 V, 20 ms, transfection of pig MSCs with green fluorescent protein (GFP) gene displayed a stable and long-term GFP expression in vitro, and untransfected pig MSCs gradually died in G418 selecting culture. Bright green fluorescence of the transfected cells could be observed under fluorescent microscopy after 24h of transfection. The gene transfection efficiency was 25%-30%. In culture of six months, the transfected cells could express high-intensive and stable GFP. Conclusions The GFP gene could be effectively expressed in pig MSCs, the transfected cells showed stable and long-term GFP expression in vitro. These results illustrated transfected MSCs expressing GFP could be used to study on differentiation of MSCs and their potential utility as cellular vehicles for systemic gene delivery for gene deal..更多还原