解剖学报

解剖学报 ›› 2021, Vol. 52 ›› Issue (2): 182-188.doi: 10.16098/j.issn.0529-1356.2021.02.004

• 神经生物学 • 上一篇    下一篇

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

王珊珊 李雯 何辉 秦建兵 田美玲 赵荷艳 成翔 金国华*   

  1. 南通大学医学院人体解剖学系，江苏省神经再生重点实验室，神经再生协同创新中心，江苏 南通 226001
  • 收稿日期:2020-08-31 修回日期:2020-10-23 出版日期:2021-04-06 发布日期:2021-04-06
  • 通讯作者: 金国华 E-mail:921930555@qq.com
  • 基金资助:
    江苏省自然科学基金青年基金

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

WANG Shanshan  LI Wen  HE Hui  QIN Jian-bing  TIAN Mei-ling  ZHAO He-yan  CHENG Xiang  JIN Guo-hua*#br#   

  1. Department of Human Anatomy, Jiangsu Key Laboratory of Nerve Regeneration, Collaborative Innovation Center for Nerve Regeneration, Medical College of Nantong University, Jiangsu Nantong 226001,China
  • Received:2020-08-31 Revised:2020-10-23 Online:2021-04-06 Published:2021-04-06
  • Contact: JIN Guo-hua E-mail:921930555@qq.com

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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。

关键词: 亨廷顿相关蛋白1, 丙戊酸钠, 神经干细胞, 神经元, 分化, 实时定量聚合酶链反应, 大鼠

Abstract:

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 marker β-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.

Key words: Huntingtin-associated protein-1, Valproate acid, Neural stem cell, Neuron, Differentiation, Real-time PCR, Rat

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