TRPM2通道对氧糖剥夺再灌注后小胶质细胞活化的影响及机制  

The Effect and Mechanism of TRPM2 Channel on the Activation of Microglia after Oxygen-glucose Deprivation/Reperfusion

作　　者：焦岩 王芳姣 和祯泉 张燕 王鹏 余建强[3] 何仲义[1] 杨巍[4] 李芳芳[4] 牛建国[1,2] JIAO Yan;WANG Fangjiao;HE Zhenquan;ZHANG Yan;WANG Peng;YU Jianqiang;HE Zhongyi;YANG Wei;LI Fangfang;NIU Jianguo(Department of Human Anatomy and Histology Embryology,School of Basic Medicine,Ningxia Medical University,Yinchuan 750004,China;Ningxia Key Laboratory of Craniocerebral Diseases,Ningxia Medical University,Yinchuan 750004,China;Department of Pharmacology,College of Pharmacy,Ningxia Medical University,Yinchuan 750004,China;Department of Biophysics,Zhejiang University School of Basic Medicine,Hangzhou 310058,China)

机构地区：[1]宁夏医科大学基础医学院人体解剖与组织胚胎学系,银川750004 [2]宁夏医科大学颅脑疾病重点实验室,银川750004 [3]宁夏医科大学药学院药理学系,银川750004 [4]浙江大学基础医学院生物物理学系,杭州310058

出　　处：《宁夏医科大学学报》2022年第7期669-676,共8页Journal of Ningxia Medical University

基　　金：宁夏重点研发计划项目(2019BFH02003)。

摘　　要：目的探究瞬时受体电位M2(transient receptor potential melastatin 2,TRPM2)通道对氧糖剥夺再灌注(oxygen-glucose deprivation/reperfusion,OGD/R)后小胶质细胞活化的影响及分子机制。方法采用线栓法建立小鼠大脑中动脉栓塞/再灌注模型(middle cerebral artery occlusion/reperfusion,MCAO/R),分为野生假手术组、TRPM2敲除假手术组、野生模型组和TRPM2敲除模型组,灌注切片,免疫荧光观察在体小胶质细胞形态。提取原代小胶质细胞,免疫荧光鉴定纯度,利用原代小胶质细胞建立(OGD/R)模型,将细胞分为野生对照组、野生模型组、野生模型+TRPM2通道抑制剂(ACA)组、TRPM2敲除对照组、TRPM2敲除模型组,使用免疫荧光观察OGD/R造模后小胶质细胞活化形态改变。在BV2细胞中建立OGD/R模型,将细胞分为空白对照组、模型组、模型+TRPM2通道抑制剂(ACA)组,利用活性氧检测试剂盒检测细胞活性氧(reactive oxygen species,ROS)水平,使用酶联免疫吸附试剂盒检测细胞炎性因子分泌情况,钙成像检测细胞胞内钙含量。结果小鼠MCAO/R造模后,野生模型组在体小胶质细胞激活显著,TRPM2敲除模型组在体小胶质细胞激活受到抑制。原代小胶质细胞OGD/R造模后,野生模型组活化形态明显,TRPM2敲除模型组、野生模型+ACA组细胞活化受到抑制。BV2细胞OGD/R造模后,与空白对照组相比,模型组ROS水平上调(P<0.05),肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素-6(interleukin-6,IL-6)分泌量增多(P<0.05)、胞内钙含量升高(P<0.05);与模型组相比,模型+ACA组的ROS水平下降(P<0.05)、TNF-α和IL-6分泌量减少(P<0.05)、胞内钙含量降低(P<0.05)。结论TRPM2通过钙离子在OGD/R中调控小胶质细胞活化及炎性因子分泌量。Objective To explore the effect and the mechanism of transient receptor potential melastain 2(TRPM2)channel on the activation of microglia under oxygen-glucose deprivation/reperfusion treatment.Methods The mouse cerebral middle cerebral artery occlusion/reperfusion(MCAO/R)model was established.by thread occlusion method.They were divided into wild type sham group,TRPM2 knockout sham group,wild type model group and TRPM2 knockout model group.The morphology of microglia in vivo was observed by perfusion section and immunofluorescence.The primary microglia were cultured and identified by immunofluorescence.The model of oxygen-glucose deprivation/reperfusion(OGD/R)was established in primary microglia.The cells were divided into 5 groups:wild type control,wild type model,wild type model+TRPM2channel inhibitor(ACA),TRPM2 knockout control and TRPM2 knockout model.Immunofluorescence was used to observe the morphological changes of microglia after OGD/R treatment.The OGD/R model was established in BV2 cells,and the cells were divided into 3 groups:control,model and model+ACA.The level of reactive oxygen species(ROS)in cells was detected by reactive oxygen species detection kit.The level of inflammatory factors was detected by enzyme-linked immunosorbent assay(Elisa)kit and the intracellular calcium was detected by calcium imaging.Results After MCAO/R modeling in mice,microglia activation in wild type model group was significant,and microglia activation in TRPM2 knockout model group was inhibited.After OGD/R modeling of primary microglia,the activated morphology of wild type model group was obvious,and the activation of TRPM2 knockout model group and wild type model+ACA group was inhibited.After OGD/R modeling of BV2 cells,compared with the control group,the level of ROS in model group was increased(P<0.05),the level of TNF-α and IL-6 in model group was increased(P<0.05),and the level of intracellular calcium in model group was increased(P<0.05).Compared with the model group,the level of ROS in model+ACA group was decreas

关 键 词：氧糖剥夺再灌注 瞬时受体电位M2 小胶质细胞活化 炎性因子 CA^(2+) 

分 类 号：R743.3[医药卫生—神经病学与精神病学]