解剖学报

解剖学报 ›› 2023, Vol. 54 ›› Issue (3): 255-260.doi: 10.16098/j.issn.0529-1356.2023.03.001

• 神经生物学 •    下一篇

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

季睿婕 李雯 金国华 张新化*
  

  1. 南通大学医学院人体解剖学系，江苏 南通 226001
  • 收稿日期:2022-12-12 修回日期:2023-01-11 出版日期:2023-06-06 发布日期:2023-06-06
  • 通讯作者: 张新化 E-mail:zhangxinhua@ntu.edu.cn
  • 基金资助:
    国家自然科学基;江苏省研究生科研创新计划项目;江苏省自然科学基金;江苏省“333”工程资助;南通市应用研究项目;中国南通大学研究生创新创业培养计划;江苏高校优势学科建设工程资助项目

Expression and effect of roundabout guidance receptor 1 during valproate induced neural stem cells differentiation

JI  Rui-jie  LI  Wen JIN  Guo-hua  ZHANG Xin-hua*   

  1. Department of Human Anatomy, Medical College of Nantong University, Jiangsu Nantong 226001, China
  • Received:2022-12-12 Revised:2023-01-11 Online:2023-06-06 Published:2023-06-06
  • Contact: ZHANG Xin-hua E-mail:zhangxinhua@ntu.edu.cn

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摘要：

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。

关键词: 回旋引导受体1, 丙戊酸钠, 神经干细胞, 分化, 神经元, 实时定量聚合酶链反应, 大鼠

Abstract:

Objective To investigate the expression and role of roundabout guidance receptor 1 (Robo1) 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 Ⅲ (Tuj1) 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 Robo1 mRNA and protein were detected by Real-time PCR and Western blotting. The dynamic changes of Robo1 mRNA were detected by Real-time PCR after the differentiation of NSCs. After the expression of Robo1 was down-regulated in NSCs by small interfering RNA, the expression of Robo1 protein was detected by Western blotting, and the expression levels of neuron-specific markers Tuj1 and microtubule associated protein-2 (MAP-2) were detected by Real-time PCR and immunofluorescence.   Results VPA induced NSCs to differentiate into neurons. Compared with the control group, the expression levels of Robo1 mRNA and protein in the differentiation of NSCs were significantly up-regulated during valproate treatment. After interference of Robo1 expression, not only Robo1 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 Robo1.

Key words: Roundabout guidance receptor 1, Valproate, Neural stem cell, Differentiation, Neuron, Real-time PCR, Rat 

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