【摘要】 目的构建脑源性神经营养因子(BDNF)基因真核表达载体,探讨BDNF过表达对神经干细胞(NSCs)向神经元分化的影响。方法采用RT-PCR法,以大鼠海马组织RNA为模板,扩增BDNF基因,定向克隆到pEGFP-N1载体中,用脂质体法转染pEGFP-N1-BDNF表达载体至NSCs中,然后用RT-PCR鉴定BDNF的表达,免疫组织化学方法鉴定NSCs向神经元的分化情况。结果成功构建了pEGFP-N1-BDNF真核表达载体,BDNF在重组质粒转染的NSCs中能够高效表达。重组质粒转染的NSCs在体外诱导分化后,能够较空质粒转染的NSCs产生更多的神经元(P<0.01)。结论 BDNF过表达能够显著促进大鼠NSCs向神经元方向分化。更多还原

【Abstract】 Objective To construct eukaryotic expression vector of brain-derived neurotrophic factor(BDNF) and detect its effect of overexpression on differentiation of rat neural stem cells(NSCs) into neurons.Methods The RT-PCR was used to amplify rat BDNF gene from RNA of rat hippocampus.The BDNF gene was inserted into eukaryotic expression vector pEGFP-N1 to construct recombinant expression vector pEGFP-N1-BDNF.The recombinant vector was transfected into NSCs by Lipofectamine 2000.The expression of BDNF mRNA in NSCs was detected by RT-PCR.The differentiation of rat NSCs into neurons was detected by immunohistochemistry staining.Results The sequence of the cloned BDNF was confirmed to be correct by DNA sequencing.The NSCs transfected with pEGFP-N1-BDNF expressed BDNF efficiently.The pEGFP-N1-BDNF transfected NSCs differentiated into more neurons than the pEGFP-N1 transfected ones(P<0.01).Conclusion All these results indicate that BDNF overexpression significantly promotes the differentiation of rat NSCs into neurons.更多还原