GFP-Nurr1基因修饰神经干细胞的建立及过表达Nurr1对神经干细胞向多巴胺神经元分化的影响

发布时间：2018-06-23 08:33

  本文选题：Nurr + 基因修饰　； 参考：《中风与神经疾病杂志》2017年05期

【摘要】：目的利用慢病毒载体建立GFP-Nurr1基因修饰的原代神经干细胞(NSCs)模型并观察Nurr1过表达后NSCs向多巴胺神经元的分化影响。方法利用基因重组构建pLenO-DCE-Nurr1慢病毒载体,用慢病毒转染第三代NSCs,转染72 h后荧光检测转染效果;设置空白对照组、空载体组及DCE-Nurr1组,分别用Western blot及PCR检测Nurr1的表达差异;并将转染后的NSCs分化培养7 d后分别用免疫细胞化学检测、Western blot及PCR检测酪氨酸羟化酶(TH)的表达。结果慢病毒转染NSCs 72 h后,转染率可达90%,与对照组相比DCE-Nurr1组高表达Nurr1。经慢病毒载体感染后的NSCs仍具备分化潜能,分化培养后发现DCE-Nurr1组分化的神经细胞中TH阳性细胞分化率90.60%,对照组为21.2%。结论慢病毒载体可高效转染NSCs过表达Nurr1;Nurr1基因过表达可以促进中脑腹侧来源NSCs向TH阳性多巴胺能神经元方向分化。
[Abstract]:Objective to establish the primary neural stem cell (NSCs) model modified by GFP-Nurr1 gene using lentivirus vector and to observe the effect of Nurr1 overexpression on the differentiation of NSCs into dopamine neurons. Methods the lentivirus vector pLenO-DCE-Nurr1 was constructed by gene recombination, the third generation of NSCs was transfected with lentivirus, the transfection effect was detected by fluorescence after 72 h, the difference of Nurr1 expression in blank control group, empty vector group and DCE-Nurr1 group was detected by Western blot and PCR, respectively. The expression of tyrosine hydroxylase (th) was detected by immunocytochemistry and Western blot. Results after 72 hours of lentivirus transfection, the transfection rate of NSCs reached 90%, and the expression of Nurr1 in DCE-Nurr1 group was higher than that in control group. NSCs infected by lentivirus vector still had differentiation potential. After differentiation and culture, it was found that the differentiation rate of th positive cells in DCE-Nurr1 group was 90.60%, and that in control group was 21.2%. Conclusion the overexpression of Nurr1N Nurr1 gene in NSCs transfected with lentivirus vector can promote the differentiation of NSCs from ventral mesencephalon into th positive dopaminergic neurons.
【作者单位】： 昆明医科大学第一附属医院神经外科;
【基金】：国家自然科学基金地区科学基金项目(No.81360197);国家自然科学基金项目(No.81241126) 云南省教育厅科学研究基金项目(No.2013C227) 云南省干细胞与再生医学重点实验室开放课题(No.201401UH00160)
【分类号】：R742.5
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本文编号：2056517