亨廷顿相关蛋白1在丙戊酸钠诱导神经干细胞分化过程中的表达和作用

Expression and role of Huntingtin-associated protein 1 during valproate induced neural stem cells differentiation

目的探讨丙戊酸钠(VPA)诱导神经干细胞(NSCs)向神经元分化过程中亨廷顿相关蛋白1(HAP-1)的表达及其作用。方法分离培养SD大鼠海马NSCs,应用Real-time PCR和Western blotting检测VPA诱导NSCs向神经元分化过程中0、1、3和5d时HAP-1 mRNA和蛋白的表达变化;Real-time PCR检测成年SD大鼠多组织及NSCs、神经元和星形胶质细胞中HAP-1 mRNA的表达;应用小干扰RNA技术下调NSCs中HAP-1的表达后,Real-time PCR和Western blotting分别检测神经元特异分子微管解聚蛋白2(Stmn-2)、神经元分化因子1(Neurod-1)、微管相关蛋白2(Map-2)和突触蛋白1(Syn-1)基因和神经元特异性标志物β-微管蛋白Ⅲ(Tuj-1)的表达水平,应用免疫荧光检测NSCs向Tuj-1阳性神经元分化的比例,并观察神经元发育情况。结果与对照组相比,VPA组NSCs向神经元分化过程中HAP-1 mRNA和蛋白在VPA处理后1 d和3 d表达明显上调;HAP-1 mRNA在中枢神经系统的海马中呈优势表达,在神经元中表达较高,NSCs中表达较低,星形胶质细胞中表达最低;干扰HAP-1表达后,VPA诱导NSCs向神经元分化的比例减少,神经元发育变差。结论VPA可能通过上调HAP-1的表达促进NSCs向神经元分化。

Objective To investigate the expression and role of Huntingtin-associated protein-1(HAP-1)in the process of valproate acid(VPA)inducing neural stem cells(NSCs)into neurons.Methods The hippocampus NSCs of SD rats were isolated and cultured,Real-time PCR and Western blotting were used to detect HAP-1 mRNA and protein expression at day 0,day 1,day 3 and day 5 during the induction of VPA on NSCs differentiation into neurons;Real-time PCR was used to detect the expression level of HAP-1 mRNA in multiple tissues of adult SD rats,as well as NSCs,neurons and astrocytes.After applying small interfering RNA technology to down-regulate the expression of HAP-1 mRNA in NSCs,Real-time PCR was used to detect the mRNA expression levels of neuron-specific molecules stathmin-2(Stmn-2),neuronal differentiation-1(Neurod-1),microtubule-associated protein-2(Map-2)and synapsin-1(Syn-1),and Western blotting was used to detect the protein expression levels of neuron-specific markerβ-tubulinⅢ(Tuj-1).Immunofluorescence was used to detect the proportion of NSCs differentiated into Tuj-1 positive neurons,and to observe the development of neurons.Results At day 1 and day 3 after VPA treatment,the expression of HAP-1 mRNA and protein in the VPA group was significantly up-regulated;HAP-1 mRNA was predominantly expressed in the hippocampus,and its expression was higher in neurons,followed by NSCs,and minimally in astrocytes.After down-regulating HAP-1 with small interference technology,the proportion of NSCs differentiated into Tuj-1 positive neurons reduced,and neuron development became worse.Conclusion VPA may promote the differentiation of NSCs into neurons by up-regulating HAP-1 expression.