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/CaMKⅡproteins 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.
基金项目
安徽省高校自然科学研究重大项目(KJ2021ZD0091)
安徽省高校自然科学研究重点项目(KJ2021A0696)
安徽省高校自然科学研究重点项目(2022AH051456)
蚌埠医学院研究生创新计划(Byycxz21013)
蚌埠医学院研究生创新计划(Byycx22008)
安徽省研究生创新创业实践项目(2022Cxcysj169)
蚌埠医学院"512人才培育计划"(by51202204)
万方数据