超顺磁氧化铁、绿色荧光蛋白双标胶质细胞源性神经营养因子基因修饰中脑神经前体细胞的建立

Establishment of SPIO and EGFP double labeled GDNF gene modified mesencephalic precursor cells

目的建立超顺磁氧化铁（SPIO）、绿色荧光蛋白（EGFP）双标胶质细胞源性神经营养因子（GDNF）基因修饰中脑神经干细胞。方法以质粒pEGFPN1-GDNF转染大鼠胚胎中脑神经干细胞，SPIO标记细胞。荧光显微镜、免疫细胞化学和Western blot鉴定EGFP、GDNF表达；普鲁土蓝染色、透射电镜鉴定SPIO标记率；诱导分化后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；免疫细胞化学、Western blot表明GDNF能正确表达；普鲁土蓝染色示SPIO标记率达100％，透射电镜示SPIO颗粒位于吞饮小泡和胞质内；体外诱导分化研究表明SPIO、EGFP双标记不影响其分化。结论成功建立SPIO、EGFP双标GDNF基因修饰中脑神经干细胞。

Objective To establish superparamagnetic iron oxide （SPIO） and enhanced green fluorescence protein （EGFP） double labeled glia cell line derived neurotrophic factor （GDNF） gene modified mesencephalic precursor cells. Methods The E14 rat embryonic mesencephalic precursor cells were isolated and cultured. The cells of the third passage were transfected with plasmid pEGFPN1-GDNF 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 under fluorescence microscope and the expression of GDNF was analyzed by immunocytochemistery, as well as Western blot. Prussian blue stain and transmission electron microscopy were used to identify the SPIO particles in cells. The distinctive marker for stem cells （ nestin ） , neuron （ β- Ⅲ -tubulin） , dopaminergic neuron （ Tyrosine Hydroxylase, TH ） , astrocytes （ GFAP ） and oligodendrocyte （CNPase） were employed to measure the differentiation ability of labeled cells. Results The expression of EGFP was initially found 12 h after transfection, increased remarkably 24 h after transfection and reached a summit at 48 h. One month after screened with medium containing G418,the positive clones were formed. Immunocytochemistery, and Western blot revealed the GDNF was expressed successfully in cells. Prussian blue stain showed numerous blue stained particles in the cytoplasma of the labeled cells. Transmission electron microscopy demonstrated vacuolar structures of different sizes under the cytoplasma within and outside of which there were highly density particles. The immunoeytochemistery, showed the labeled cells were nestin positive, and after differentiation the cells expressed GFAP, CNPase, β-Ⅲ-tubulin and TH. Conclusion The SPIO and EGFP double labeled GDNF gene modified mesencephalic precursor cells were successfully established, which are multipotent and have the ability to self-renew and will provide the foundation for the further research about cell therapy of Parkinson＇ s disease.