文章摘要
Wnt/β-catenin信号通路在体外调控诱导性多能干细胞向神经干细胞分化中的作用及机制研究
The Role and Mechanism of Wnt/β-catenin Signaling Pathway in Regulating the Differentiation of Induced Pluripotent Stem Cells into Neural Stem Cells in Vitro
投稿时间:2020-11-30  修订日期:2021-04-23
DOI:
中文关键词: 诱导性多能干细胞,神经分化,神经干细胞,Wnt/β-catenin信号通路,Wnt3a,DKK-1,IWR-1
英文关键词: induced pluripotent stem cells  neural differentiation  neural stem cells  Wnt/β-catenin signaling pathway  Wnt3a  DKK-1  IWR-1
基金项目:国家自然科学基金(31570994)
作者单位邮编
周丽萍 浙江中医药大学生命科学学院 310053
林德菊 浙江中医药大学生命科学学院 
周斌杰 浙江中医药大学生命科学学院 
姚盼盼 浙江中医药大学生命科学学院 
余勤 浙江中医药大学生命科学学院 310053
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中文摘要:
      [目的] 探讨Wnt/β-catenin信号通路在体外调控诱导性多能干细胞(Induced pluripotent stem cell,iPS细胞)向神经干细胞分化中的作用及机制。[方法] 体外培养小鼠iPS细胞,采用N2B27培养基联合RA诱导法,诱导其向神经干细胞分化;免疫荧光法检测iPS细胞来源神经干细胞中的Nestin、βIII Tubulin的表达情况;将细胞分为正常对照组、Wnt3a组、Dickkopf-1(DKK-1)组和IWR-1组,采用Real-time qPCR、Western blot分别检测iPS细胞神经分化过程中,Nestin、βIII Tubulin及β-catenin的表达情况;采用悬浮培养法检测Wnt/β-catenin信号通路对iPS细胞来源神经干细胞增殖能力的影响。[结果] iPS细胞分化来源神经干细胞表达神经干细胞的表面特异性标志物Nestin及早期的神经元特异性标志物βIII Tubulin;在iPS细胞向神经干细胞分化过程中,Nestin、βIII Tubulin的表达均提高(P<0.05,P<0.05),β-catenin表达降低(P<0.05);采用Wnt3a处理后,iPS细胞来源神经干细胞增殖能力较弱;采用DKK-1和IWR-1处理后, iPS细胞来源神经干细胞增殖能力较强,且Nestin表达提高(P<0.01),β-catenin表达降低(P<0.05)。[结论] Wnt/β-catenin信号通路在iPS细胞向神经干细胞诱导分化过程中受抑制;采用DKK-1和IWR-1下调Wnt/β-catenin信号通路,可促进iPS细胞向神经干细胞分化,提高iPS细胞来源神经干细胞增殖能力,提示Wnt/β-catenin信号通路在iPS细胞向神经干细胞分化过程中具有负调控的作用。
英文摘要:
      [Objective] To explore the role and mechanism of the Wnt/β-catenin signaling pathway in regulating differentiation of induced pluripotent stem (iPS) cells into neural stem cells in vitro. [Methods] Mouse iPS cells were induced to differentiate into neural stem cells by N2B27 medium and RA after their cultivation in vitro. Immunofluorescence was utilized to determine the expression of Nestin and βIII tubulin in iPS cells-derived neural stem cells. The cells were then divided into normal Control group, Wnt3a group, Dickkopf-1 (DKK-1) group and IWR-1 group. Real time qPCR and Western blot were emploved to detect the expression of Nestin, βIII tubulin and β-catenin in the neural differentiation process of iPS cells. The effects of Wnt/β-catenin signaling pathway on the proliferation of iPS cells-derived neural stem cells were detected by suspension culture. [Results] The iPS cells could be induced to form neural stem cells that expressed Nestin and βIII Tubulin. In the precess of neural differentiation, the expression of Nestin and βIII tubulin were increased (P<0.05, P<0.05), and the expression of β-catenin, a key factor of Wnt/β-catenin signaling pathway, was decreased (P<0.05). The proliferation of iPS cells-derived neural stem cells treated by Wnt 3a was weak. However, after DKK-1 and IWR-1 treatment, the proliferation of iPS cell-derived neural stem cells was strong, and the expression of Nestin was increased (P<0.01), the expression of β-catenin was decreased (P<0.05). [Conclusion] Wnt/β-catenin signaling pathway was inhibited during the differentiation of iPS cells into neural stem cells. The down-regulation of Wnt/β-catenin signaling pathway by DKK-1 and IWR-1 can promote the differentiation of iPS cells into neural stem cells and improve the proliferation of iPS cells derived neural stem cells. It is suggested that Wnt/β-catenin signaling pathway plays a negative role in the neuronal differentiation of iPS cells.
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