中性粒细胞对永久缺血性脑卒中小胶质细胞极化的调控作用  

Regulatory effect of neutrophils in microglial polarization after permanent ischemic stroke

作　　者：黄敏铧 叶欣妍 吴思雨 罗少潼 吴芷珊 陈元 平苏宁 HUANG Min-hua;YE Xin-yan;WU Si-yu;LUO Shao-tong;WU Zhi-shan;CHEN Yuan;PING Su-ning(Neurobiology Research Center,School of Medical,Sun Yet-sen University,Shenzhen 518107,China)

机构地区：[1]中山大学医学院神经生物研究中心,深圳518107

出　　处：《解剖学报》2025年第2期136-142,共7页Acta Anatomica Sinica

基　　金：国家自然科学基金青年基金(8230062128);广东省自然科学基金(2023A1515010374);深圳市基础研究项目(JCYJ20220530145615034)。

摘　　要：目的在永久缺血性脑卒中小鼠模型上,探讨外周血中性粒细胞浸润对大脑原位小胶质细胞的极化调控。方法取58只C57BL/6小鼠,分为假手术组和模型组,对模型组小鼠实施永久性大脑中动脉阻塞手术。术后48 h、7 d、14 d和30 d处死小鼠,灌注取材。Western blotting检测脑组织中M1型小胶质细胞标志物CD16、M2型标志物精氨酸酶1(Arg1)、炎症因子白细胞介素-1β(IL-1β)及中性粒细胞标志物髓过氧化物酶(MPO)的水平。免疫荧光法检测脑切片梗死区周围中性粒细胞浸润及M2型小胶质细胞分布。在体外实验中,将分离纯化的中性粒细胞与小胶质细胞BV2共培养,脂多糖刺激后,观察BV2吞噬中性粒细胞的情况,并检测共培养后BV2细胞CD16和Arg1蛋白的表达水平。结果Western blotting显示,小鼠术后脑组织中CD16(P<0.05)、IL-1β(P<0.001)和MPO(P<0.05)的水平在48 h和7 d显著增高,随后下降,MPO于术后30 d恢复正常。免疫荧光显示,小鼠大脑皮层梗死区周围的MPO阳性细胞在术后48 h显著增加(P<0.001),随后降低(P<0.05)。小胶质细胞标志物离子钙结合适配分子1(Iba1)和MPO双阳性细胞在术后逐渐增多,14 d达到最高(P<0.05),Iba1和Arg1双阳性细胞也在术后7 d(P<0.05)和14 d(P<0.01)显著上升。体外共培养显示,BV2吞噬中性粒细胞后,CD16显著下降(P<0.05),Arg1显著上调(P<0.05)。结论在永久缺血性脑卒中小鼠模型中,小胶质细胞吞噬中性粒细胞后从M1型向M2型转变,损伤脑区由促炎转变为抗炎状态。Objective To investigate the effects of peripheral blood neutrophil infiltration on the polarization regulation of cerebral resident microglia under a permanent ischemic stroke model.Methods Fifty⁃eight C57BL/6 mice were divided into two groups.One group was sham group,and the other group of mice was subjected to permanent middle cerebral artery occlusion surgery.Mice were euthanized 48 hours,7 days,14 days,and 30 days after surgery for tissue collection.Western blotting was used to detect expression levels of M1 microglia markers CD16,M2 microglia marker arginase 1(Arg1),inflammatory cytokine interleukin⁃1β(IL⁃1β),and neutrophil marker myeloperoxidase(MPO)in brain tissue.Immunofluorescence histochemical staining was used to assess neutrophil infiltration and M2 microglial distribution around the infarct area in brain sections.In vitro,purified neutrophils were co⁃cultured with BV2 microglial cells.After lipopolysaccharide stimulation,the phagocytosis of neutrophils by BV2 cells was observed,and the expression levels of CD16 and Arg1 proteins in BV2 cells were detected.Results Western blotting showed that the levels of CD16(P<0.05),IL⁃1β(P<0.001),and MPO(P<0.05)in brain tissue increased significantly 48 hours and 7 days after surgery,then decreased,with MPO expression returning to normal levels 30 days after surgery.Immunofluorescence showed a significant increase of MPO⁃positive cells around the infarct area of the mouse cerebral cortex 48 hours after surgery(P<0.001),followed by a decrease(P<0.05).The number of ionized calcium binding adapter molecule 1(Iba1)and MPO double⁃positive cells gradually increased after surgery,and reached their peak at 14 days(P<0.05).Iba1 and Arg1 double⁃positive cells also increased significantly 7 days(P<0.05)and 14 days(P<0.01)after surgery.In vitro,co⁃culture experiments showed that after BV2 phagocytosing neutrophils,CD16(P<0.05)significantly decreased and Arg1 significantly upregulated(P<0.05).Conclusion In a permanent ischemic stroke model,microglia trans

关 键 词：缺血性脑卒中 小胶质细胞 中性粒细胞 M1/M2极化 神经炎症 免疫印迹法 免疫荧光 C57BL/6小鼠 

分 类 号：R328[医药卫生—人体解剖和组织胚胎学] R743[医药卫生—基础医学]