摘要
目的探讨调节性T细胞(Tregs)减轻脑出血(intracerebral hemorrhage,ICH)所致的炎症损伤的具体机制。方法 (1)将20只C57BL/6小鼠按照随机数字表法分为ICH Tregs治疗组、ICH对照组和假手术Tregs治疗组、假手术对照组,每组5只,采用自体血建立小鼠ICH模型,尾静脉注射Tregs,对照组注射等量生理盐水,4 d后检测各组小鼠神经功能障碍评分、脑组织含水量、血肿体积变化,ELISA检测血肿周围组织中IL-6、TNF-α、IL-10、TGF-β表达改变,PCR检测血肿周围组织CCL3、iNOS、Arg1、Ym1表达改变,组织免疫荧光双染CD16/32和CD206。(2)构建BV2/Tregs transwell共培养体系,用LPS/IL-4激活BV2细胞,共培养72 h后ELISA检测IL-6、TNF-α、IL-10、TGF-β表达改变,PCR检测CCL3、iNOS、Arg1、Ym1表达改变。IL-10抗体中和IL-10,Western blot检测STAT3、磷酸化STAT3、TGF-β表达,PCR检测Arg1、Ym1表达。结果在动物实验中,Tregs治疗后神经功能障碍评分、含水量、血肿体积均减少(P<0.05),IL-6、TNF-α、CCL3、iNOS表达减少(P<0.05),IL-10、TGF-β、Arg1、Ym1表达增加(P<0.05),血肿周围组织M2型小胶质细胞占总小胶质细胞百分比增高(P<0.05)。在细胞实验中,Tregs共培养组IL-6、TNF-α、CCL3、iNOS表达减少(P<0.05),IL-10、TGF-β、Arg1、Ym1表达增加(P<0.05),pSTAT3表达增加(P<0.05)。IL-10被中和后,与Tregs共培养组比较,pSTAT3蛋白、TGF-β蛋白表达量及Arg1、Ym1 mRNA表达量减少(P<0.05)。结论 Tregs可能通过IL-10/STAT3信号通路诱导激活的小胶质细胞向M2型极化,从而减轻ICH所致的炎症损伤。
Objective To investigate the effect of regulatory T cells（ Tregs） on inflammatory injury induced by intracerebral hemorrhage（ ICH） and explore the molecular mechanism. Methods Twenty C57 BL/6 mice were randomized into 4 equal groups,namely ICH with Tregs treatment group,ICH model group,sham operation group,and sham operation with Tregs treatment group. Mouse models of ICH were established by injecting non-anti-coagulated autologous blood（ 20 μL） into the basal ganglia,followed 24 h later by injection of autologous Tregs via the tail vein（ normal saline was injected in ICH model and sham operation groups）. The changes in neurological deficit score,brain tissue water content,and hematoma volume were observed in the mice 4 days after ICH. ELISA was used to detect the levels of interleukin-6（ IL-6）,tumor necrosis factor-α（ TNF-α,） IL-10,and transforming growth factor-β（ TGF-β） in the brain tissues,and PCR was used for detecting the changes in the mRNA expression of the signature genes（ CCL3,iNOS,Arg1,and Ym1）; immunofluorescence assay was used for detecting the specialized protein markers M1（ CD16/32） and M2（ CD206）. We also established a BV2 cells/Tregs transwell co-culture system,in which the same inflammatory cytokines and mRNA expressions of the signature genes were detected; Western blotting was used to detect the protein expression of STAT3,p-STAT3 and TGF-β in the co-culture. Results In mice with ICH,intravenous delivery of Tregs significantly improved the neurological function,relieved cerebral edema and reduced the hematoma volume（ P〈0. 05）; administration of Tregs also significantly lowered the expression levels of IL-6,TNF-α,CCL3,and iNOS and increased the levels of IL-10,TGF-β,Arg1,and Ym1 in the brain tissues（ P〈0. 05）. In the co-culture of Tregs and BV2 cells,the expression levels of IL-6,TNF-α,CCL3,iNOS and p-STAT3 increased and those of IL-10,TGF-β,Arg1,and Ym1 decreased significantly as compared with those in the control group（ P〈0. 05）. Neutralization of IL-10 in the co-culture caused significant reductions in the expression levels of p-STAT3 and TGF-β proteins and Arg1 and Ym1 mRNA（ P〈0. 05）. Conclusion Tregs alleviate ICH-induced inflammatory injury in mice possibly by modulating microglia M2 polarization through the IL-10/STAT3 pathway.
作者
李秋萌
张文倩
谭赢
赵贯建
张翔
张鹏
程远
LI Qiumeng;ZHANG Wenqian;TAN Ying;ZHAO Guanjian;ZHANG Xiang;ZHANG Peng;CHENG Yuan(Department of Neurosurgery;Department of Gynecology and Obstetrics,the Second Affiliated Hospital of Chongqing Medical University,Chongqing,400010,China)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2018年第16期1461-1468,共8页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(81371674
81401505)~~
