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目的探讨核受体相关因子1(Nurr1)基因对大鼠中脑神经干细胞(NSCs)体外诱导分化为多巴胺能神经元的作用。方法通过基因重组技术构建pEGFP-N1-Nurr1真核表达载体,用电穿孔法将重组质粒转染第3代NSCs,用荧光显微镜观察转染效率,免疫印迹法检测基因表达效果;并对转染后的NSCs进行体外分化培养3周,用免疫荧光双标技术检测酪氨酸羟化酶(TH)和微管相关蛋白-2(MAP-2)的表达。结果电穿孔法转染NSCs48h后转染效率达35%,1周后免疫印迹法检测到Nurr1蛋白高表达,约为对照组的5倍;Nurr1基因转染的NSCs分化为TH阳性神经元的比率为15.45%,明显高于对照组,而MAP-2阳性神经元数量与对照组相似。结论 Nurr1基因过表达可以诱导NSCs向TH阳性的多巴胺能神经元分化。
Objective To investigate the effect of nuclear receptor related factor 1 (Nurr1) gene on the differentiation of rat neural stem cells (NSCs) into dopaminergic neurons in vitro. Methods The eukaryotic expression vector pEGFP-N1-Nurr1 was constructed by gene recombination technique. The third generation NSCs were transfected with the recombinant plasmid by electroporation. The transfection efficiency was observed by fluorescence microscopy and the gene expression was detected by Western blotting. The NSCs were cultured in vitro for 3 weeks. The expression of tyrosine hydroxylase (TH) and microtubule-associated protein-2 (MAP-2) were detected by double immunofluorescence staining. Results The transfection efficiency of NSCs was 35% after electroporation for 48 hours. The expression of Nurr1 protein was detected by immunoblotting in 1 week, which was about 5 times that of the control group. The percentage of Nurr1 transfected NSCs differentiated into TH-positive neurons Was 15.45%, significantly higher than the control group, while the number of MAP-2 positive neurons was similar to the control group. Conclusion Overexpression of Nurr1 can induce NSCs to differentiate into TH-positive dopaminergic neurons.