神经干细胞的体外培养及增强型绿色荧光蛋白基因转染研究

目的探索大鼠胚胎神经干细胞(NSCs)的体外培养,探讨NSCs作为基因靶细胞,以及逆转录病毒介导增强型绿色荧光蛋白(EGFP)标记NSCs的可行性.方法胚胎大鼠脑组织分离神经干细胞,无血清培养,免疫组织化学技术鉴定.用Lipofectin将pLEGFP-N1逆转录病毒载体导入PA317包装细胞,用G418筛选获得抗性细胞克隆,扩增抗性细胞并收集病毒上清;病毒上清感染神经干细胞,用G418筛选,荧光显微镜下观察并挑选EGFP表达最强的克隆,并做免疫组化鉴定;进一步培养扩增,做传代细胞的分化鉴定.结果培养出大鼠胚胎神经干细胞.荧光显微镜下检测感染逆转录病毒的神经干细胞,以及免疫组化鉴定,均显示EGFP获得良好表达;而且感染细胞能够被诱导分化为神经元和神经胶质细胞.结论大鼠胚胎神经干细胞能够在体外适宜的条件下进行长期培养;神经干细胞可直接作为基因靶细胞;利用逆转录病毒载体,建立稳定表达EGFP的神经干细胞系具有可行性,为进一步做标记细胞移植研究奠定基础.

A Study on Culture of Neural Stem Cells in vitro and Transfection of Enhanced Green Fluorescent Protein

Objective To explore the culture of neural stem cells (NSCs) in vitro and the feasibility of NSCs being gene target cells and enhanced green fluorescent protein (EGFP) labeling NSCs. Methods The NSCs were isolate from rat embryo brains, then cultured in serum free medium, and identified by immunocytochemistry. pLEGFP-N1 retroviral vectors were transferred into PA317 cells by lipofectin. After selected by G418, the resistant colonies were cloned, and the virus supernatant was collected. The NSCs were infected by the virus supernatant, and then selected by G418. The colonies with the best expression of EGFP were observed and selected using fluorescence microscope, and identified with immunocytochemistry technology, which were further cloned and their passage cells were also identified with immunocytochemistry technology. Results The NSCs were successfully cultured and G418 resistant clones of NSCs gave off strikingly bright green fluorescence. The EGFP expressed well, and affected cells could be induced to differentiate into neurons and glial cells respectively. Conclusions The NSCs originated from rat embryo brains can be cultured in vitro under appropriate conditions, and it can be directly used as gene target cells. Moreover, it is feasible to construct the NSCs expressing EGFP stably with the use of retroviral vectors, which can be further applied for the transplantation study of labeling cells.