回旋引导受体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 effect of roundabout guidance receptor 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回旋引导受体1(Robo1)的表达及作用。方法分离培养SD大鼠海马NSCs,正常NSCs和应用VPA处理NSCs各提取自10只SD大鼠。应用VPA处理后,免疫荧光技术检测NSCs向神经元标志物β-微管蛋白Ⅲ(Tuj1)阳性神经元分化的比例;利用基因芯片技术检测正常NSCs和VPA处理后NSCs中差异表达mRNA并进行生物信息学分析;通过Real-time PCR、Western blotting检测VPA诱导NSCs分化过程中Robo1 mRNA和蛋白的表达情况;Real-time PCR检测诱导分化后NSCs中Robo1 mRNA的动态变化;应用小干扰RNA下调NSCs中Robo1,Western blotting检测Robo1蛋白的表达情况,Real-time PCR和免疫荧光技术检测NSCs中神经元特异性标志物Tuj1和微管相关蛋白2(MAP-2)表达情况。结果应用VPA处理NSCs可促进其向神经元分化;VPA处理组与对照组相比,Robo1 mRNA和蛋白的表达显著上调;NSCs诱导分化过程中Robo1表达水平逐渐上升;干扰Robo1表达后,VPA诱导NSCs分化过程中Robo1上调受到抑制,VPA诱导NSCs向神经元分化的比例减少。结论VPA或通过上调Robo1的表达促进NSCs向神经元分化。

Objective To investigate the expression and role of roundabout guidance receptor 1(Robol)in the neuronal differentiation of neural stem cells(NSCs)induced by valproate(VPA).Methods The hippocampus NSCs of SD rats were isolated and cultured.Normal NSCs and VPA-treated NSCs were extracted from 10 SD rats.After VPA treatment,the proportion of neuron-specific marker β-tubulin II(Tujl)positive neurons differentiated from NSCs were detected by immunofluorescence.The differentially expressed mRNA in normal NSCs and VPA-induced NSCs were detected by gene chip technology.After VPA treatment,the expression levels of Robol mRNA and protein were detected by Real-time PCR and Western blotting.The dynamic changes of Robol mRNA were detected by Real-time PCR after the differentiation of NSCs.After the expression of Robol was down-regulated in NSCs by small interfering RNA,the expression of Robol protein was detected by Western blotting,and the expression levels of neuron-specific markers Tujl and microtubule associated protein-2(MAP-2)were detected by Real-time PCR and immunofluorescence.Result VPA induced NSCs to differentiate into neurons.Compared with the control group,the expression levels of Robol mRNA and protein in the differentiation of NSCs were significantly up-regulated during valproate treatment.After interference of Robol expression,not only Robol upregulation was inhibited during the differentiation of NSCs induced by VPA,but also the proportion of NSCs differentiated into neurons decreased.Conclusion VPA may promote the differentiation of NSCs into neurons by up-regulating the expression of Robol.