摘要
目的探讨基质相互作用分子1(STIM1)对脑缺血再灌注损伤后小胶质/巨噬细胞M1型活化的影响及机制。方法(1)动物实验:采用随机数字表法将20只雄性C57BL/6J小鼠分为假手术组、大脑中动脉缺血再灌注(MCAO/R)组、MCAO/R+si-Ctrl组及MCAO/R+si-STIM1组,后3组小鼠建立MCAO/R模型,MCAO/R+si-Ctrl组及MCAO/R+si-STIM1组小鼠分别转染空载体对照病毒和STIM1基因敲除慢病毒。观察STIM1转染效率及各组小鼠中小胶质/巨噬细胞M1型活化标记物分化抗原86(CD86)的表达情况。(2)细胞实验:将原代小胶质细胞分为Ctrl组、氧糖剥夺/复氧(OGD/R)组、OGD/R+si-Ctrl组、OGD/R+si-STIM1组、OGD/R+溶媒组及OGD/R+4-苯基丁酸(4-PBA)组。后5组细胞构建OGD/R模型,OGD/R+si-Ctrl组、OGD/R+si-STIM1组细胞分别转染空载体对照病毒和STIM1基因敲除慢病毒;OGD/R+4-PBA组细胞在OGD/R造模前24 h使用1 mmol/L预处理1 h以抑制内质网应激(ERS),OGD/R+溶媒组细胞同时间使用0.5%二甲基亚砜(DMSO)预处理1 h。采用Western blotting、ELISA、RT-qPCR等方法检测细胞模型中小胶质/巨噬细胞M1型活化标记物CD86、炎性因子肿瘤坏死因子-α(TNF-α)mRNA、IL-1β及ERS相关蛋白[转录因子C/EBP同源蛋白(CHOP)、活化转录因子4(ATF4)]的表达情况。结果(1)动物实验:MCAO/R+si-STIM1组STIM1表达水平较假手术组、MACO/R组及MCAO/R+si-Ctrl组均明显降低,差异均有统计学意义(P<0.05)。与MCAO/R组及MCAO/R+si-Ctrl组比较,MCAO/R+si-STIM1组小鼠缺血灶周围CD86与Iba-1共表达的小胶质/巨噬细胞数显著降低,差异有统计学意义(P<0.05)。(2)细胞实验:与OGD/R组及OGD/R+si-Ctrl组相比,OGD/R+si-STIM1组细胞STIM1、CD86、TNF-αmRNA表达水平及上清液中IL-1β含量均显著降低,差异均有统计学意义(P<0.05)。同样地,与OGD/R组及OGD/R+si-Ctrl组相比,OGD/R+si-STIM1组细胞ERS相关蛋白ATF4、CHOP表达水平亦显著降低,差异均有统计学意义(P<0.05)。此外,与OGD/R组及OGD/R+溶
Objective To investigate the influence and mechanism of stromal interaction molecule 1(STIM1)in microglia/macrophages M1 activation after cerebral ischemia-reperfusion injury.Methods(1)Animal experiment:20 male C57BL/6J mice were randomly divided into sham-operated(Sham)group,middle cerebral artery occlusion and reperfusion(MCAO/R)group,MCAO/R+si-Ctrl group,and MCAO/R+si-STIM1 group(n=5);MCAO/R models were established in mice of the latter 3 groups;empty vector control virus and STIM1 gene knockout lentivirus were transfected into mice in the MCAO/R+si-Ctrl group and MCAO/R+si-STIM1 group.The transfection efficiency of STIM1 and the expression of microglia/macrophages M1 activation marker cluster of differentiation 86(CD86)in each group were observed.(2)Cell experiment:primary microglia were divided into Ctrl group,oxygen-glucose deprivation/re-oxygenation(OGD/R)group,OGD/R+si-Ctrl group,OGD/R+si-STIM1 group,OGD/R+solvent group,and OGD/R+4-phenylbutyric acid(4-PBA)group;OGD/R models were established in the later 5 groups;empty vector control virus and STIM1 gene knockout lentivirus were transfected into mice in the OGD/R+si-Ctrl group and OGD/R+si-STIM1 group;cells in the OGD/R+4-PBA group were pre-treated with 1 mmol/L 4-PBA for 1 h at 24 h before OGD/R modelling to inhibit endoplasmic reticulum stress(ERS),and cells in the OGD/R+solvent group were pre-treated with 0.5%dimethyl sulfoxide(DMSO)for 1 h at the same time.Reverse transcription quantitative polymerase chain reaction(RT-qPCR),ELISA,Western blotting and other methods were used to detect the levels of CD86,tumour necrosis factor-α(TNF-α)mRNA,interleukin(IL)-1β,and ERS-related proteins(transcription factor C/EBP homologous protein[CHOP],activated transcription factor 4[ATF4])in these cells.Results(1)Animal experiment:the STIM1 expression in MCAO/R+si-STIM1 group was significantly lower than that in Sham group,MACO/R group and MCAO/R+si-Ctrl group(P<0.05);as compared with that in the MACO/R group and MCAO/R+si-Ctrl group,the number of microglia/macropha
作者
谢文宇
张洪晨
鲁传豪
冯元
张磊
吕超
时全星
戴舒惠
李侠
Xie Wenyu;Zhang Hongchen;Lu Chuanhao;Feng Yuan;Zhang Lei;Lyu Chao;Shi Quanxing;Dai Shuhui;Li Xia(Department of Neurosurgery,First Affiliated Hospital of Air Force Medical University,Xi'an 710032,China;Department of Neurosurgery,Strategic Support Force Characteristic Medical Center of People's Liberation Army,Beijing 100101,China)
出处
《中华神经医学杂志》
CAS
CSCD
北大核心
2022年第8期762-769,共8页
Chinese Journal of Neuromedicine
基金
国家自然科学基金(81974188)
陕西省重点研发计划项目(2019SF-032)
陕西省创新能力支撑计划(2022TD-42)。
