M2型巨噬细胞高表达乳脂球表皮生长因子8促进脐静脉内皮细胞增殖和迁移  

The high expression of milk fat globule epidermal growth factor factorⅧin M2 macrophages promoted the proliferation and migration of human umbilical vein endothelial cells

作　　者：邝莉雯 李恭驰[2] 张晨晨 申利 王知 李炳辉[1,3] Kuang Liwen;Li Gongchi;Zhang Chenchen;Shen Li;Wang Zhi;Li Binghui(Department of Wound Prosthodontics Center,Liyuan Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430077,China;Department of Hand Surgery,Union Hospital affiliated to Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430022,China;Hubei Clinical Research Center of Chronic Wound and Diabetic Podiatry,Wuhan 430077,China;Department of Obstetrics and Gynecology,Liyuan Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430077,China)

机构地区：[1]华中科技大学同济医学院附属梨园医院创面修复中心,武汉430077 [2]华中科技大学同济医学院附属协和医院手外科,武汉430022 [3]湖北省慢性创面及糖尿病足医学临床研究中心,武汉430077 [4]华中科技大学同济医学院附属梨园医院妇产科,武汉430077

出　　处：《中华实验外科杂志》2022年第12期2344-2348,共5页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金青年科学基金项目(81801922);国家重大疾病多学科合作诊疗能力建设项目(国卫办医函[2019]542号);湖北省自然科学基金面上项目(2020CFB696);湖北省科技创新平台专项资助建设项目(2018B1CC340);湖北省重点研发计划项目(2020BCB029)。

摘　　要：目的探讨不同亚型巨噬细胞条件培养基对脐静脉内皮细胞(HUVECs)的生物学功能影响及机制。方法将巨噬细胞RAW264.7分为M0、M1、M2和si-乳脂球表皮生长因子-8(MFG-E8)组。M0型巨噬细胞不做处理;M1型巨噬细胞采用脂多糖(LPS,100 ng/ml)及γ-干扰素(IFN-γ,10 ng/ml)联合刺激;M2型巨噬细胞使用白细胞介素(IL)-4(10 ng/ml)诱导;si-MFG-E8组通过小干扰寡核苷酸(siRNA)沉默MFG-E8的表达。诱导48 h后免疫荧光法鉴定巨噬细胞极化,实时定量聚合酶链反应(RT-PCR)验证siRNA敲降效率,并收集各组巨噬细胞条件培养基;酶联免疫吸附试验(ELISA)检测各组巨噬细胞及其条件培养基中MFG-E8的表达;细胞计数试剂盒(CCK-8)检测各组巨噬细胞条件培养基对脐静脉内皮细胞增殖能力的影响;Transwell实验检测各组巨噬细胞条件培养基对脐静脉内皮细胞迁移能力的影响;RT-PCR检测各组的磷脂酰肌醇3激酶(PI3K)、蛋白激酶B(Akt)和雷帕霉素靶蛋白(mTOR)分子的表达水平。组间比较采用非配对t检验。结果免疫荧光法鉴定巨噬细胞成功极化为M1和M2型。RT-PCR验证与si-NC比较,si-MFG-E8组MFG-E8 mRNA的表达含量明显降低(t=63.070,P<0.01)。ELISA实验结果显示,与M0组比较,M2组巨噬细胞MFG-E8明显增高,M1及si-MFG-E8组明显降低(t_(02)=14.050,P_(02)<0.01;t_(01)=28.120,P_(01)<0.01;t_(0si)=48.690,P_(0si)<0.01);与M0组比较,M2组巨噬细胞条件培养基中MFG-E8明显增高,M1及si-MFG-E8组明显降低(t_(02)=17.240,P_(02)<0.01;t_(01)=16.350,P_(01)<0.01;t_(0si)=36.260,P_(0si)<0.01)。CCK-8实验结果显示,与M0组比较,经M2型巨噬细胞条件培养基处理的HUVECs的增殖能力明显增加,M1及si-MFG-E8组明显下降(t_(02)=7.563,P_(02)<0.01;t_(01)=4.365,P_(01)<0.01;t_(0si)=5.965,P_(0si)<0.01)。Transwell实验结果显示,与M0组比较,经M2型巨噬细胞条件培养基处理的HUVECs的迁移能力明显增加,M1及si-MFG-E8组明显下降,(t_(02)=5.339,P_(02)<0.05;t_(01)=2.991,P_(0Objective To investigate the effect of conditioned medium of different subtypes of macrophages on the biological function of human umbilical vein endothelial cells(HUVECs)and its mechanism.Methods Macrophages RAW264.7 were divided into M0,M1,M2 and si-milk fat globule epidermal growth factor factorⅧ(MFG-E8)groups.M0 macrophages were not treated specifically;M1 macrophages were stimulated with LPS(100 ng/ml)and IFN-γ(10 ng/ml);M2 macrophages were induced with IL-4(10 ng/ml);si-MFG-E8 group’s expression of MFG-E8 was silenced by short interfering oligonucleotide(siRNA).After 48 h of induction,macrophage polarization was identified by immunofluorescence,siRNA knockdown efficiency was verified by real-time quantitative polymerase chain reaction(RT-PCR),and the conditioned medium was collected from each group of macrophages.The expression of MFG-E8 in each group of macrophages and their conditioned media was measured by enzyme linked immunosorbent assay(ELISA);the effect of macrophage conditioned media on the proliferation of umbilical vein endothelial cells was measured by cell counting kit-8(CCK-8);the effect of macrophage conditioned media on the migration ability of HUVECs was measured by Transwell assay;the expression levels of phosphatidylinositol 3 kinase(PI3K),protein kinase B(Akt)and mammalian target of rapamycin(mTOR)molecules in each group were measured by RT-PCR.Unpaired t-test was used for comparison between groups.Results Macrophages were successfully polarized into M1 and M2 by immunofluorescence assay.Compared with si-NC,the expression of MFG-E8 mRNA in the si-MFG-E8 group was significantly decreased(t=63.070,P<0.01).ELISA results showed that compared with the M0 group,the expression of MFG-E8 in M2 group was significantly increased,M1 and the si-MFG-E8 group were significantly decreased(t_(02)=14.050,P_(02)<0.01;t_(01)=28.120,P_(01)<0.01;t_(0si)=48.690,P_(0si)<0.01);Compared with group M0,MFG-E8 in conditioned medium of macrophages in group M2 was significantly increased,while M1 and si-MFG-E8 we

关 键 词：巨噬细胞 脐静脉内皮细胞 条件培养基 乳脂球表皮生长因子-8 

分 类 号：R392.12[医药卫生—免疫学]