摘要
目的建立绿色荧光蛋白（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12h后EGFP开始表达；普鲁士蓝染色显示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。
Objective To establish enhanced green fluorescence protein （EGFP） and superparamagnetic iron oxide （SPIO） double labeled mesencephalic neural stem cells. Methods The E14 rat embryonic mesencephalic neural stem cells were isolated and cultured. The cells of the third passage were transfected with plasmid pEGFPN1 using FuGENE HD transfection reagent and screened with medium containing G418. The positive clones were selected, proliferated and then labeled with SPIO 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） , dopaminergic neuron （ Tyrosine 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 transfection, 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-tnbulin-Ⅲ and TH. Conclusion The EGFP and SPIO double labeled E14 rat embryonic mesencephalic neural stem cells were established.
出处
《中华神经外科杂志》
CSCD
北大核心
2008年第10期787-790,共4页
Chinese Journal of Neurosurgery
基金
国家自然科学基金（30672151）
关键词
绿色荧光蛋白
超顺磁氧化铁
中脑神经干细胞
大鼠
胚胎
Green fluorescent protein
Superparamagnetic iron oxide
Mesencephalie neural stem cells
Rats,embryo
