TRPM2对小鼠脑缺血再灌注损伤中星形胶质细胞活化的影响  

The Effect of TRPM2 on the Activation of Astrocytes in Cerebral Ischemia-reperfusion in Mice

作　　者：王芳姣 焦岩 和祯泉 张燕 张瑞 余建强[3] 何仲义[1] 杨巍[4] 李芳芳[4] 牛建国[1,2] WANG Fangjiao;JIAO Yan;HE Zhenquan;ZHANG Yan;ZHANG Rui;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;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 Schoolof 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689-695,共7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)在小鼠脑缺血再灌注(ischemia-reperfusion,I/R)过程中对星形胶质细胞活化的影响。方法 在体实验:设置野生假手术(WT-sham)组、TRPM2敲除假手术(TRPM2^(-/-)-sham)组、野生脑缺血再灌注(WT-I/R)组、TRPM2敲除脑缺血再灌注(TRPM2^(-/-)-I/R)组,采用“线栓法”建立小鼠大脑中动脉I/R模型,采用Bederson评分方法评价小鼠神经功能缺损情况;2,3,5-氯化三苯基四氮唑(2,3,5-triphenyltetrazolium chloride,TTC)染色评价小鼠脑梗死体积;免疫荧光染色观察小鼠缺血侧海马区及皮层缺血半暗带区星形胶质细胞的活化情况。离体实验:纯化培养原代星形胶质细胞并建立氧糖剥夺再灌注(oxygen and glucose deprivation-reperfusion,OGD/R)模型,Western blot检测各组细胞胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)表达情况。结果 在体实验中,与WT-sham组相比,WT-I/R组小鼠Bederson评分较高(P<0.05),脑梗死体积较大(P<0.05),缺血侧海马CA1、CA3、DG区及皮层缺血半暗带区GFAP荧光强度及阳性细胞数量增多(P<0.05);与WT-I/R组相比,TRPM2-/--I/R组小鼠Bederson评分降低(P<0.05),脑梗死体积减小(P<0.05),缺血侧GFAP荧光强度及阳性细胞数量显著减少(P<0.05)。离体实验中,与WT-CTRL(control,CTRL)组相比,WT-OGD/R组细胞GFAP蛋白表达量增高(P<0.05);与WT-OGD/R组相比,TRPM2^(-/-)-OGD/R组GFAP蛋白表达量降低(P<0.05)。结论 TRPM2敲除可抑制星形胶质细胞活化从而降低脑I/R损伤。Objective To investigate the effect of transient receptor potential melastatin 2(TRPM2) on the activation of astrocytes during cerebral ischemia-reperfusion(I/R) in mice.Methods In vivo experiments:wild type(WT-sham),TRPM2 knockout(TRPM2^(-/-)-sham),WT-I/R and TRPM2^(-/-)-I/R groups were set and the mouse cerebral I/R model were established.Bederson score was used to evaluate the neurological function of mice;2,3,5-triphenyltetrazolium chloride(TTC)staining was used to evaluate the cerebral infarction rate in mice;Immunofluorescence staining was used to observe the activation of astrocytes in the ischemic hippocampus and the ischemic penumbra of the cortex.In vitro experiments:Primary astrocytes were purified and cultured to establish oxygen and glucose deprivation-reperfusion(OGD/R)model,and then Western blot was used to detect the expression of GFAP protein in each group.Results Compared with WT-sham group,WT-I/R group had higher Bederson score(P < 0.05)and larger cerebral infarction volume(P<0.05),besides,both the GFAP fluorescence intensity and the number of positive cells in the ischemic hippocampal CA1,CA3,DG region and cortical ischemic penumbra(P<0.05)were significantlyincreased(P<0.05).Compared with WT-I/R group,TRPM2^(-/-)-I/R group had lower Bederson score(P<0.05),lesser cerebral infarction volume(P<0.05),and reduced GFAP fluorescence intensity and the decreased number of positive cells on the ischemic side(P<0.05).In vitro experiments showed that compared with WT-CTRL(control,CTRL)group,the expression of GFAP protein in WT-OGD/R group was increased(P<0.05).Compared with WT-OGD/R group,GFAP protein expression in TRPM2^(-/-)-OGD/Rgroupwas decreased(P<0.05).Conclusion TRPM2 knockout can significantly inhibit astrocyte activation and reduce cerebral ischemia-reperfusion injury(CIRI).

关 键 词：瞬时受体电位M2 脑缺血再灌注损伤 星形胶质细胞 氧糖剥夺再灌注 胶质纤维酸性蛋白 

分 类 号：R743.3[医药卫生—神经病学与精神病学]