缺氧特异性诱导肺血管内皮细胞黏附分子3表达以促进单核细胞黏附  

Hypoxia specifically induces CADM3 expression in pulmonary endothelial cells and promotes monocyte adhesion

作　　者：孟祥琼 陈婷 谢宏晨 杨诚忠 范旭 谭小玲[1,2] MENG Xiangqiong;CHEN Ting;XIE Hongchen;YANG Chengzhong;FAN Xu;TAN Xiaoling(Department of Cold Region Medicine,Faculty of High Altitude Military Medicine,Army Medical University(Third Military Medical University),Chongqing;Key Laboratory of Extreme Environmental Medicine of Ministry of Education,Faculty of High Altitude Military Medicine,Army Medical University(Third Military Medical University),Chongqing;Department of Plateau Physiology and Pathology,Faculty of High Altitude Military Medicine,Army Medical University(Third Military Medical University),Chongqing;Center for Reproductive Medicine,Chongqing Health Center for Women and Children(Women and Children Hospital of Chongqing Medical University),Chongqing;Medical School,Hubei Minzu University,Enshi,Hubei,China)

机构地区：[1]陆军军医大学(第三军医大学)高原军事医学系寒区医学教研室,重庆 [2]陆军军医大学(第三军医大学)高原军事医学系极端环境医学教育部重点实验室,重庆 [3]陆军军医大学(第三军医大学)高原军事医学系高原生理学与病理学教研室,重庆 [4]重庆市妇幼保健院(重庆医科大学附属妇女儿童医院)生殖医学中心,重庆 [5]湖北民族大学医学部,湖北恩施

出　　处：《陆军军医大学学报》2025年第1期51-59,共9页Journal of Army Medical University

基　　金：国家自然科学基金面上项目(32071122)。

摘　　要：目的探究细胞黏附分子3(cell adhesion molecular 3,CADM3)在肺血管内皮细胞上的表达特异性,分析其介导单核细胞特异性黏附的作用,探讨缺氧诱导肺血管外周单核细胞浸润的新机制。方法以人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)、大鼠肺血管内皮细胞(rat pulmonary vascular endothelial cell,rPEC)和大鼠主动脉内皮细胞(rat aortic endothelial cell,rAEC)为细胞模型,分为常氧(21%O2)对照组和缺氧(1%O2或5%O2)处理组。通过分析缺氧8 h HUVEC的转录组数据,筛选差异表达的CADM3。通过细胞黏附实验、siRNA干扰、免疫细胞化学技术、Western blot技术和流式细胞术,分析CADM3在缺氧HUVEC与单核细胞特异性高黏附中的作用;通过铁螯合剂-去铁胺(deferoxamine,DFX)和shRNA干扰调节HIF-1α表达,分析HIF-1转录活性对CADM3表达的影响。结果缺氧HUVEC中CADM3表达增加,在缺氧6~8 h达到最高,随后下降;靶向CADM3的siRNA转染后,与阴性对照组比较,CADM3的表达降低,缺氧HUVEC和U937细胞的黏附率显著下降,差异具有统计学意义(P<0.05)。与常氧(21%O2)溶剂对照组比较,DFX(100μmol/L)处理的HUVEC中HIF-1α及其靶蛋白STC2蛋白水平增加;靶向HIF-1α的shRNA转染后,与阴性对照组比较,HIF-1α及其靶蛋白STC2蛋白水平下降,但CADM3蛋白表达均无变化。缺氧rPEC中CADM3的蛋白水平及其在细胞膜上的分布均增加,与rPEC细胞比较,rAEC细胞中未检测到CADM3的蛋白表达;脂多糖(lipopolysaccharide,LPS)(5μg/mL)处理后,常氧或缺氧培养的rPEC细胞中HIF-1α和CADM3表达均无明显变化。缺氧rPEC与外周血白细胞中CD11b+细胞的黏附比率最高,差异具有统计学意义(P<0.01)。抗CADM3抗体孵育后,与Blank组比较,缺氧rPEC与U937细胞的黏附显著下降,差异具有统计学意义(P<0.05)。结论缺氧以特异地非HIF-1依赖的方式诱导肺血管内皮细胞中CADM3的表达,促进缺氧诱导的肺血管内皮细胞与单核细胞的Objective To investigate the expression profile of cell adhesion molecular 3(CADM3)on pulmonary endothelial cells,analyze its role in mediating specific adhesion to monocyte,and explore a new mechanism of hypoxia inducing peripheral monocyte infiltration in pulmonary vessels.Methods Human umbilical vein endothelial cells(HUVEC),rat pulmonary vascular endothelial cells(rPEC),and rat aortic endothelial cells(rAEC)were subjected,and divided into normoxic(21%O2)control group and hypoxic(1%O2 or 5%O2)treatment group.Analyzing the transcriptome data of HUVEC exposure to hypoxia for 8 h screened a differentially expressed molecule,CADM3.Cell adhesion experiments,siRNA interference,immunohistochemical assay,Western blotting,and flow cytometry were used to study the role of CADM3 in hypoxia specific high adhesion of HUVEC monocytes.Iron chelator deferoxamine(DFX)and shRNA interference were employed to enhance the expression of HIF-1α,and the effect of HIF-1 transcriptional activity on CADM3 expression was analyzed.Results Hypoxic treatment resulted in enhanced expression of CADM3 in HUVEC,and the expression level reached the peak at 6-8 h after hypoxia,and then decreased.Transfection with siRNA targeting CADM3 decreased the expression of CADM3,and significantly reduced the adhesion rate of hypoxic HUVEC-U937 cells when compared with the negative control group(P<0.05).Compared with the solvent control group,the protein levels of HIF-1αand its target protein STC2 in HUVEC treated with DFX(100μmol/L)were increased.Transfection with shRNA targeting HIF-1αled the protein levels of HIF-1αand its target protein STC2 decreased,but had no effect on the protein expression of CADM3 in comparison to the negative control group.The protein level and distribution of CADM3 on the cell membrane were increased in hypoxic rPEC.No expression of CADM3 protein was found in the rAEC when compared with rPEC.After treatment with 5μg/mL LPS,there were no significant changes in HIF-1α,STC2 and CADM3 in rPEC cultured under normoxia or hypoxia

关 键 词：缺氧 细胞黏附 肺血管内皮细胞 单核细胞 细胞黏附分子3 

分 类 号：R322.12[医药卫生—人体解剖和组织胚胎学] R331.142[医药卫生—基础医学] R364.4