摘要目的 建市绿色荧光蛋白(EGFP)、超顺磁氧化铁(SPIO)双标大鼠胚胎中脑神经干细胞.方法 以质粒pEGFPN1转染大鼠胚胎中脑神经干细胞后用SPIO标记.荧光显微镜观察EGFP表达情况;普鲁士蓝染色、透射电镜鉴定SPIO标记;体外诱导分化,免疫细胞化学鉴定其分化能力.结果 基因转染12 h后EGFP开始表达;普鲁十蓝染色显示SPIO标记满意,透射电镜显示SPIO位于吞饮小泡和胞质内;体外诱导分化研究表明EGFP、SPIO双标不影响其增殖与分化.结论 成功建立了EGFP、SPIO双标大鼠胚胎中脑神经干细胞.

abstractsObjective To establish enhanced green fluorescence protein (EGFP) and superparamagnetic iron oxide (SPIO) double labeled mesencephalie neural stem cells. Methods The El4 rat embryonic mesencephalic neural stem cells were isolated and cultured. The cells of the third passage were transfected with plasmid pEGFPN! using FuGENE HD transfection reagent and screened with medium containing G418. The positive clones were selected,proliferated and then labeled with SP10 mediated by FuGENE HD transfection reagent. The expression of EGFP was observed with fluorescence microscope.Prussian blue stain and transmission electron microscopy were used to identify the SPIO particles in cells.The distinctive markers for neuron (β-tubulin-III ),dopaminergie neuron ( Tyrasine Hydroxylase,TH ),astrocyte (GFAP) and oligodendrocyte (CNPase) were employed to detect the phenoltype of differentiated cells. Results The expression of EGFP was initially found 12 hours after transfeetion,increased remarkably 24 hours after transfection and reached a peak value at 48 hours. One month after screened with medium containing G418,positive clones were formed. Prussian blue stain showed numerous blue stained particles in the cytoplasma of the labeled cells. Transmission electron microscopy showed vacuolar structures of different sizes under the cytoplasma, within and outside which there were high density particles. The immunocytochemistery showed the labeled cells were Nestin positive,after differentiation the cells expressed GFAP,CNPase,β-tubulin-III and TH. Conclusion The EGFP and SPIO double labeled El4 rat embryonic mesencephalic neural stem cells were established.