蛋白酶活化受体1在热打击人脐静脉内皮细胞炎症激活中作用的研究  

Study on the role of protease-activated receptor 1 in the inflammatory activation of human umbilical vein endothelial cells by heat stress

作　　者：王郑莲 徐秋林 邓茂林 刘亚楠[4] 苏磊 Wang Zhenglian;Xu Qiulin;Deng Maolin;Liu Yanan;Su Lei(The Department of Emergency,Changsha Hospital of Traditional Medicine(Changsha Eighth Hospital),Changsha,Hunan 410100,China;Department of Pathophysiology,Guangdong Provincial Key Laboratory of Shock and Microcirculation,School of Basic Medical Sciences,Southern Medical Uni-versity,Guangzhou,Guangdong 510515,China;Department of Emergency and Critical Medicine,Guangdong Provincial People’s Hospital,Guangdong Academy of Medical Science,Guangzhou,Guangdong 510000,China;Department of ICU,Nanfang Hospital,Guangzhou,Guangdong 523326,China;Department of ICU,Southern Theater Command General Hospi-tal,Guangzhou,Guangdong 510010,China)

机构地区：[1]长沙市中医医院本部急诊科,湖南长沙410100 [2]南方医科大学生命科学楼病理生理实验室,广东广州510515 [3]广东省人民医院重症医学科,广东广州510000 [4]南方医科大学南方医院重症医学科,广东广州523326 [5]南部战区总医院重症医学科,广东广州510010

出　　处：《感染、炎症、修复》2023年第3期173-179,F0002,F0003,共9页Infection Inflammation Repair

基　　金：广东省自然科学基金博士启动项目(S2013040015661);中国博士后科学基金项目(2014M552180)。

摘　　要：目的:观察热打击对人脐静脉内皮细胞(HUVECs)中蛋白酶活化受体1(PAR1)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、细胞间黏附分子-1(ICAM-1)的mRNA及蛋白表达水平的影响,探讨PAR1在热打击后HUVECs炎症激活中的作用。方法:培养的HUVECs分为37℃对照组(37℃培养)和42℃热打击组(42℃培养2 h后再于37℃继续培养至观察时间点)。42℃热打击细胞再分别预先采用PAR1抑制剂SCH79797(42℃+SCH组)、激活剂TFLLR-NH2(42℃+TFL组)、PAR1 siRNA转染(42℃+PAR1siRNA组)、腺病毒转染(42℃+AdPAR1)等预处理,再行热打击。各组细胞PAR1、TNF-α、IL-6、ICAM-1的mRNA表达水平采用逆转录-聚合酶链反应(RT-PCR)方法检测,PAR1蛋白水平采用蛋白质免疫印迹试验(Western Blot)检测,TNF-α、IL-6、ICAM-1的蛋白表达水平采用酶联免疫吸附法(ELISA)检测。将人单核细胞THP1与各组HUVECs共同培养,倒置荧光显微镜下观察单核细胞对HUVECs的黏附情况。结果:与37℃对照组比较,42℃热打击组PAR1、TNF-α、IL-6、ICAM-1的mRNA及蛋白表达水平在热打击结束后显著增高(P<0.05或P<0.01);siRNA转染能有效降低而腺病毒转染能有效升高常温培养及热打击后HUVECs的PAR1蛋白表达(P<0.01)。与单纯热打击HUVECs比较,SCH预处理及PAR1siRNA转染能有效降低HUVECs热打击后TNF-α、IL-6、ICAM-1的mRNA及蛋白水平,并能有效减轻单核细胞对HUVECs热打击后的黏附作用;TFL、AdPAR1能有效增加HUVECs热打击后TNF-α、IL-6、ICAM-1的mRNA及蛋白表达水平,并能有效增强单核细胞对HUVECs热打击后的黏附(P<0.05,P<0.01)。结论:热打击可导致HUVECs的PAR1活化,PAR1参与了热打击HUVECs炎症激活的过程。Objective:To observe mRNA and protein expression level of protease-activated receptor 1(PAR1),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),intercellula cell adhesion molecule-1(ICAM-1)after heat sress(HS)of human umbilical vein endothelial cells(HUVECs),and to study the role of PAR1 in inflammtory responses of HUVECs by heat stress.Methods:HUVECs were pretreated with PAR1 inhibitor SCH79797(SCH),PAR1 agonist TFLLR-NH2(TFL),PRA1siRNA and PAR1 adenovirus respectively and then reeeived HS(42℃2 h),different groups were extracted to detect mRNA and protein expression of PAR1,TNF-α,IL-6,and ICAM-1.Mononuclear cells adhesion to HUVECs was observed by fluorescence microscopy after pre-treated with PAR1 inhibitor,agonist,siRNA and PAR1 adenovirus respectively in HUVECs in 8 hours after heat stress.Results:Control with normal group,mRNA and protein expressions of PAR1,TNF-α,IL-6 and ICAM-1 increased after heat stress(P<0.05,P<0.01);PAR1 protein expression of HUVECs after high normal temperature culture and heat stress could be effectively decreased by siRNA transfection and increased by adenovirus transfection(P<0.01).Control with HS group,inhibiting the expression of PAR1 or PRA1siRNA lead to TNF-α,IL-6,and ICAM-1 mRNA and pro⁃tein level decreasing and the adhesion of monocytes decreasing(P<0.05,P<0.01);otherwise,increasing PAR1 expression or AdPAR1 lead to TNF-α,IL-6,and ICAM-1 mRNA and protein level increasing and the adhesion increasing(P<0.05,P<0.01).Conclusions:HS can lead to PAR1 activation in HUVECs,which leads to the activation of inflammatory pathways in HUVECs.

关 键 词：热打击 人脐静脉内皮细胞 蛋白酶活化受体1 炎症反应 单核细胞 

分 类 号：R594.12[医药卫生—内科学] R34[医药卫生—临床医学]