CD137信号调控巨噬细胞M1/M2极性转变促进血管新生  

CD137 signaling promotes angiogenesis through regulating macrophage M1/M2 polarization

作　　者：耿田欣 李波[1] 许尧 王中群[1] 邵晨[1] 严金川[1] Geng Tianxin;Li Bo;Xu Yao;Wang Zhongqun;Shao Chen;Yan Jinchuan(Department of Cardiology,Affiliated Hospital of Jiangsu University,Zhenjiang 212001,China)

机构地区：[1]江苏大学附属医院心内科,镇江212001

出　　处：《中华心血管病杂志》2020年第6期500-506,共7页Chinese Journal of Cardiology

基　　金：国家自然科学基金(81970379,81670405);江苏省自然基金(BK20161355);镇江市心血管病临床医学研究中心(SS2018008)。

摘　　要：目的探讨CD137-CD137L信号(简称CD137信号)是否通过调控巨噬细胞M1/M2极性转变促进血管新生。方法将3%巯基乙酸盐肉汤诱导的小鼠腹腔原代巨噬细胞分3组,即对照组、CD137信号激动组和CD137信号抑制组,通过检测M1和M2型巨噬细胞的相关特异性标志物观察巨噬细胞表型的变化,采用流式细胞术(FCM)检测巨噬细胞表面CD137、CD86及CD206的表达,Western blot和RT-PCR检测巨噬细胞诱导性一氧化氮合酶(iNOS)、Ⅰ型精氨酸酶(Arg-1)的蛋白和mRNA表达。酶联免疫吸附测定法(ELISA)检测巨噬细胞培养上清中白细胞介素(IL)-12和IL-10分泌水平。将巨噬细胞和内皮细胞(bEnd.3)共培养,上室种植巨噬细胞,下室基质胶种植内皮细胞,实验分为3组,即对照组、CD137信号激动组和过氧化物酶体增殖物激活受体-γ(PPAR-γ)抑制组,检测各组内皮细胞的管腔形成能力。结果(1)分离的小鼠腹腔原代巨噬细胞纯度为(97.93±1.31)%,巨噬细胞表面CD137表达为(97.40±2.70)%。(2)与对照组比较,CD137信号激动组Arg-1 mRNA和蛋白表达水平较高(P<0.05),iNOS mRNA和蛋白表达水平较低(P<0.05);与CD137信号激动组比较,CD137信号抑制组Arg-1 mRNA和蛋白表达水平较低(P<0.05),iNOS mRNA和蛋白表达水平较高(P<0.05)。流式细胞术显示,CD137信号激动组CD206平均荧光强度高于对照组(P<0.05),而CD86平均荧光强度低于对照组(P<0.01);CD137信号抑制组CD206表达明显低于CD137信号激动组(P<0.05),CD86表达高于CD137信号激动组(P<0.01)。ELISA显示,CD137信号激动组IL-10分泌高于对照组(P<0.01),IL-12分泌明显低于对照组(P<0.01);而与CD137信号激动组比较,CD137信号抑制组IL-10分泌较低(P<0.05),IL-12分泌较高(P<0.05)。(3)内皮管腔形成实验显示,CD137信号激动组内皮细胞小管长度大于对照组,分支数量多于对照组(P<0.05);PPAR-γ抑制组的内皮细胞小管长度及分支数量的形成明显被抑制(P<0.05)。结论CD137信号�Objective To investigate whether CD137 signaling can promote angiogenesis via regulating macrophage M1/M2 polarization.Methods(1)The primary peritoneal macrophages in mice induced by 3%thiglycollate broth were divided into three groups:control group,CD137 signaling activated group and CD137 signaling inhibited group.Various specific markers of M1 and M2 macrophages were detected to observe the phenotype change of macrophages,and the macrophages protein expression of CD137,CD86 and CD206 was detected by flow cytometry(FCM).The protein and mRNA expression of induced nitric oxide synthase(iNOS),arginaseⅠ(Arg-1)was determined by Western blot and RT-PCR,respectively.The secretion levels of IL-12 and IL-10 in culture supernatant of macrophages were detected by ELISA.(2)Macrophages were co-cultured with the endothelial cells(bEnd.3),and macrophages were implanted in the upper chamber,endothelial cells were implanted in stromal glue of the lower chamber.The experiment was divided into three groups:the control group,CD137 signaling activated group and peroxisome proliferator-activated receptor-γ(PPAR-γ)inhibited group,and tube formation ability of endothelial cells in each group was determined.Results(1)The purity of primary peritoneal macrophages in mice was(97.93±1.31)%.The expression of CD137 on the surface of macrophages was(97.40±2.70)%.(2)Compared with control group,the mRNA and protein expression levels of Arg-1 were significantly increased and the mRNA and protein expression of iNOS were significantly decreased in CD137 signaling activated group(all P<0.05).Compared with CD137 signaling activated group,the mRNA and protein expression of Arg-1 were significantly lower and the mRNA and protein expression levels of iNOS were significantly higher in CD137 signaling inhibited group(all P<0.05).FCM results showed that the average fluorescence intensity of CD206 was higher,while the average fluorescence intensity of CD86 was lower in CD137 signaling activated group than in control group(P<0.05,P<0.01,respectively);

关 键 词：动脉粥样硬化 巨噬细胞极化 CD137 血管新生 

分 类 号：R54[医药卫生—心血管疾病]