文章摘要
周丽萍,林德菊,周斌杰,等.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[J].浙江中医药大学学报,2021,45(8):809-815.
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 Cell into Neural Stem Cell in Vitro
DOI:10.16466/j.issn1005-5509.2021.08.001
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 cell  neural differentiation  neural stem cell  Wnt/β-catenin signaling pathway  Wnt3a  DKK-1  IWR-1
基金项目:国家自然科学基金项目(31570994)
作者单位E-mail
周丽萍 浙江中医药大学生命科学学院 杭州 310053  
林德菊 浙江中医药大学生命科学学院 杭州 310053  
周斌杰 浙江中医药大学生命科学学院 杭州 310053  
姚盼盼 浙江中医药大学生命科学学院 杭州 310053  
余勤 浙江中医药大学生命科学学院 杭州 310053 qinyu3587@126.com 
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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)向神经干细胞分化中的作用及机制。[方法]体外培养小鼠iPS,采用N2B27培养基联合维甲酸(retinoic acid,RA)诱导法,诱导其向神经干细胞分化;免疫荧光法检测iPS来源神经干细胞中的巢蛋白(Nestin)、β-微管蛋白Ⅲ(β-tubulinⅢ)的表达情况;将细胞分为正常组、Wnt3a组、Dickkopf相关蛋白-1(Dickkopf-1,DKK-1)组和IWR-1组,采用实时荧光定量聚合酶链式反应(Real-time quantitative polymerase chain reaction,Real-time qPCR)、Western blot分别检测iPS神经分化过程中Nestin、β-tubulin Ⅲ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来源神经干细胞增殖能力的影响。[结果]iPS来源神经干细胞表达神经干细胞的表面特异性标志物Nestin,以及早期神经元特异性标志物β-tubulin Ⅲ;iPS向神经干细胞分化过程中,Nestin、β-tubulin Ⅲ的表达均升高(P<0.05,P<0.05),β-catenin表达降低(P<0.05);采用Wnt3a处理后,iPS来源神经干细胞增殖能力较弱;采用DKK-1和IWR-1处理后,iPS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向神经干细胞诱导分化过程中受到抑制;采用DKK-1和IWR-1下调Wnt/β-catenin信号通路,可促进iPS向神经干细胞分化,提高iPS来源神经干细胞的增殖能力,提示Wnt/β-catenin信号通路在iPS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 the differentiation of induced pluripotent stem cell(iPS) into neural stem cell in vitro.[Methods] Mice iPS were induced to differentiate into neural stem cell by N2B27 medium and retinoic acid(RA) after cultivated in vitro. Immunofluorescence was utilized to detect the expression of Nestin and β-tubulin Ⅲ in iPS-derived neural stem cells. The cells were then divided into normal group, Wnt3a group, Dickkopf-1(DKK-1) group and IWR-1 group. Real-time quantitative polymerase chain reaction(Real-time qPCR) and Western blot were employed to detect the expression of Nestin, β-tubulin Ⅲ and β-catenin in the neural differentiation process of iPS. The effects of Wnt/β-catenin signaling pathway on the proliferation of iPS-derived neural stem cells were detected by suspension culture.[Results]The iPS-derived neural stem cells express Nestin, a surface specific marker of neural stem cells, and β-tubulin Ⅲ, an early neuron specific marker. In the process of neural differentiation, the expression of Nestin and β-tubulin Ⅲ were increased(P<0.05, P<0.05), and the expression of β-catenin was decreased(P<0.05). The proliferation of iPS-derived neural stem cells was weak after treated by Wnt3a. However, after DKK-1 and IWR-1 treatment, the proliferation of iPS-derived neural stem cells enhanced, 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 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 into neural stem cells and improve the proliferation of iPS-derived neural stem cell, which suggests that Wnt/β-catenin signaling pathway plays a negative role in the neuronal differentiation of iPS.
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