【摘要】 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。更多还原

【Abstract】 Objective To study the effect of orphan nuclear receptor related factor 1(Nurr1) gene on inducing neural stem cells(NSCs) derived from rat midbrain to differentiate into dopaminergic neurons in vitro.Methods The eukaryotic expression vector pEGFP-N1-Nurr1 was constructed by gene recombination technology and transfected into P3 NSCs through electroporation.The transfection efficiency was measured by fluorescence microscope,and the expression of Nurr1 in NSCs was detected by Western blotting.After differentiated for 3 weeks in vitro,the expression of tyrosine hydroxylase(TH) and microtubule associated proteins-2(MAP-2) were detected by double-immunofluorescence labeling.Results The transfection efficiency was about 35% 48 hours after transfection by electroporation,and Nurr1 protein was over-expressed 1 week after transfection with recombinant pEGFP-N1-Nurr1,increased about 5 times compared with the control group.Immunofluorescence showed that the proportion of TH positive neurons was 15.45%,which was markedly increased as compared with the control group.But there was no difference with the number of MAP-2 positive neurons.Conclusion The findings demonstrate that NSCs can be induced to differentiate into TH positive dopaminergic neurons through Nurr1 gene overexpression.更多还原