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    氧糖剥夺再灌注对神经元Wnt5a/FZD2/Ca2+通路的影响

    The impact of oxygen-glucose deprivation/reperfusion on the Wnt5a/FZD2/Ca2+ pathway in neurons

      摘要
    • 摘要:
      目的 探讨氧糖剥夺再灌注(OGD/Rep)后神经元细胞内Wnt5a/FZD2/Ca2+通路的变化。
      方法 采用OGD/Rep模型构建神经元细胞局部缺血/再灌注损伤，分正常组、再灌注(0、3、6、9、12、24 h)组。免疫荧光鉴定皮层神经元细胞，CCK-8法确定神经元细胞OGD时间，Western blotting检测非经典Wnt/Ca2+信号通路中Wnt5a、FZD2、IP3-R和p-CaMKⅡ/CaMKⅡ蛋白的变化, 免疫荧光和酶标仪检测细胞内Ca2+和活性氧(ROS)的含量。
      结果 MAP-2免疫荧光鉴定原代皮层神经元细胞纯度较高。与正常组比较，神经元细胞OGD 3 h后细胞存活率为55.46%；与正常组比较，OGD/Rep后非经典Wnt/Ca2+信号通路相关蛋白Wnt5a(3、6、9、12、24 h)、FZD2(3、6、9 h)、IP3-R(6、9、12、24 h)和p-CaMKⅡ/CaMKⅡ(6 h)的相对表达量上调(P＜0.05~P＜0.01)；与正常组比较，OGD/Rep后神经细胞内Ca2+和ROS的表达量均升高(P＜0.01)。
      结论 神经元细胞OGD/Rep能激活Wnt5a/FZD2/Ca2+信号通路。

       

      Abstract:
      Objective  To investigate the changes in the Wnt5a/FZD2/Ca2+pathway in neuronal cells after oxygen glucose deprivation reperfusion (OGD/Rep).
      Methods  The OGD/Rep model was used to construct neuronal cell local ischemia/reperfusion injury, and cells were divided into normal group and reperfusion (0, 3, 6, 9, 12, 24 h) group.Cortical neuron cells was identified by immunofluorescence.The OGD time was determined by CCK-8 method.Western blotting was used to detect the changes of Wnt5a, FZD2, IP3-R, and p-CaMKⅡ/CaMKⅡ proteins in non-classical Wnt/Ca2+ signaling pathways.Immunofluorescence and enzyme-linked immunosorbent assay (ELISA) were used to detect the levels of intracellular Ca2+ and ROS.
      Results  MAP-2 immunofluorescence identification showed that the purity of primary cortical neurons was relatively high.Compared with the normal group, the survival rate of neuronal cells after 3 hours of OGD was 55.46%.Compared with the normal group, the relative expression levels of non classical Wnt/Ca2+ signaling pathway related proteins Wnt5a (3, 6, 9, 12, 24 h), FZD2 (3, 6, 9 h), IP3-R (6, 9, 12, 24 h), and p-CaMKⅡ/CaMKⅡ (6 h) were upregulated after OGD/Rep (P < 0.05 to P < 0.01).Compared with the normal group, the expression levels of Ca2+ and ROS in nerve cells increased after OGD/Rep (P < 0.01).
      Conclusions  OGD/Rep in neuronal cells can activate the Wnt5a/FZD2/Ca2+signaling pathway.

       

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