BDNF、EGFP基因修饰大鼠胚胎腹侧中脑神经干细胞的建立

Establishment of Midbrain-Derived Neural Stem Cells Modified by BDNF and EGFP Gene

目的建立脑源性神经营养因子(BDNF)、增强型绿色荧光蛋白(EGFP)基因修饰大鼠胚胎腹侧中脑神经干细胞(mNSCs)。方法以FuGENE HD转染试剂介导质粒pcDNA3-BDNF、pEGFPN1共转染第三代E14大鼠胚胎mNSCs。荧光显微镜观察EGFP表达情况;免疫细胞化学方法和Western blot鉴定BDNF的表达;体外诱导分化后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;免疫细胞化学、Western blot结果表明BDNF能在细胞中正确表达;体外诱导分化研究表明BDNF、EGFP基因修饰不影响大鼠胚胎mNSCs的增殖与分化。结论成功建立了BDNF、EGFP基因修饰大鼠胚胎mNSCs,可为进一步开展帕金森病的细胞移植治疗研究奠定基础。

Objective To establish midbrain-derived neural stem ceils （mNSC） modified by brain--derived neurotrophic factor （BDNF） and enhanced green fluorescent protein （EGFP） gene. Methods The embryonic mNSC of El4 rat were isolated and cultured. The cells of the third passage were co-transfected with plasmid pcDNA3-BDNF and pEGFPN1 by using FuGENE HD transfection reagent. The transfected ceils were screened with medium containing G418. The positive clones were selected and proliferated. The expression of EGFP was observed under a fluorescence microscope. The expression of BDNF was analyzed by immunocytochemistry as well as western blot. The distinctive markers for neuron （β-Ⅲ-tubuhn）, dopaminergic neuron （Tyrosine Hydroxylase, TH）, astrocyte （glial fibrillay acidic protein, GFAP） and oligodendrocyte （cyclic nucleotide 3＇phosphohydrolose, CNPase） were employed to detect the type of the differentiated cells. Results The expression of EGFP was initially found 12 hours after the transfection, increased significantly 24 hours after the transfection and reached a summit 48 hours after the transfection. One month after screening with medium containing G418, the positive clones were formed. The immunocytochemistery and western blot showed that the BDNF was expressed successfully in the cells. The ceils modified by BDNF and EGFP gene were positive Nestin. After the differentiation, the cells expressing GFAP, CNPase, β- Ⅲ-tubulin and TH respectively were observed. Conclusion The embryonic mNSC of E14 rat by modified the BDNF and EGFP gene were successfully established. The mNSC, which can be differentiated into dopaminergic neurons and have the ability to self-renewal, will provide the foundation for the further research on the therapy of Parkinson＇s disease.