NGF-β、GFP基因修饰大鼠胚胎中脑神经干细胞的建立

Proliferation and differentiation of NGF-β and GFP gene modified midbrain-derived neural stem cells

目的建立神经生长因子β(NGF-β)基因修饰大鼠胚胎中脑神经干细胞。方法以质粒pcDNA3-hNGFb、pEGFPN1共转染大鼠胚胎中脑神经干细胞,荧光显微镜观察绿色荧光蛋白(GFP)在细胞内的表达,免疫细胞化学、Western blot鉴定NGF-β在细胞内的表达。体外诱导分化,免疫细胞化学鉴定其分化能力。结果GFP在基因转染12h后开始表达,免疫细胞化学、Western blot结果表明NGF-β能在细胞中正确表达且NGF-β、GFP基因修饰不影响其增殖与分化。结论成功建立NGF-β、GFP基因修饰大鼠胚胎中脑神经干细胞。

Objective To establish nerve growth factor beta （NGF-β） and green fluorescent protein （GFP） gene modified midbrain-derived neural stem cells. Methods The E14 rat embryonic midbrain-derived neural stem ceils were isolated and cultured. The cells of the third passage were eo-transfected with plasmid pcDNA3- hNGFb and pEGFPN1 using FuGENE HD transfection reagent. The expression of GFP was observed with fluorescence microscope. Forty-eight hours after transfeetion, the transfected ceils were screened with medium containing C,418. The positive clones were selected, proliferated and then identified with immunocylochemistry for nestin and the expression Of NGF-β was analyzed by immunocytochemistry as well as Western blot. At the same time, the ceils were induced to differentiation. The distinctive marker for neuron （β-Ⅲ-tubulin）, dopaminergie neuron （tyrosine hydroxylase, TH）, astrocyte （glial fibrillary acidieprotein, GFAP） were employed to detect the phenol type of differentiated cells. Results The expression of GFP 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. The immunocytochemistry and Western blot showed the NGF-β was expressed successfully in cells. In addition, the immunocytochemistry showed the cells were nestin positive, and the cells expressed β-Ⅲ-tubulin, TH and GFAP after differentiation. Conclusion The NGF-β and GFP gene modified E14 rat embryonic midbrain-derived neural stem cells are 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.