血管紧张素Ⅱ/乳脂肪球表皮生长因子8信号通路调控人脐静脉内皮细胞基质重构的作用及分子机制  

Effect of angiotensinⅡ/milk fat globule epidermal growth factor 8 signaling on matrix remodeling in human umbilical vein endothelial cells and underlying molecular mechanism

作　　者：倪冷[1] 狄潇[1] 张锐 马百涛 刘昌伟[1] 郑曰宏[1] Ni Leng;Di Xiao;Zhang Rui;Ma Baitao;Liu Changwei;Zheng Yuehong(Department of Vascular Surgery,Chinese Academy of Medical Sciences,Peking Union Medical College,Peking Union Medical College Hospital,Beijing 100730,China)

机构地区：[1]中国医学科学院北京协和医学院北京协和医院血管外科,100730

出　　处：《中华实验外科杂志》2021年第1期53-56,共4页Chinese Journal of Experimental Surgery

基　　金：中央高校基本科研业务费专项资金(3332019028)。

摘　　要：目的探讨血管紧张素Ⅱ(AngⅡ)/乳脂肪球表皮生长因子8(MFG-E8)信号通路在人脐静脉内皮细胞(HUVECs)基质重构调控中的分子生物学机制。方法利用实时定量聚合酶链反应(RT-qPCR)实验对AngⅡ和MFG-E8浓度进行筛选,根据实验需求在AngⅡ和MFG-E8浓度筛选实验中分为5组,阴性对照组(DPBS)、AngII 0.1μmol/L、AngII 1.0μmol/L、MFG-E850.0μg/L、MFG-E8100.0μg/L。并利用RT-qPCR和蛋白质印迹法(Western blot)检测AngⅡ和MFG-E8的相互作用关系及对下游基因单核细胞趋化蛋白-1(MCP-1)、基质金属蛋白酶-2(MMP-2)和转化生长因子-β1(TGF-β1)表达的影响。两组间比较采用t检验,多组间比较采用单因素方差分析,组间两两比较采用SNK-q检验。结果在脐静脉内皮细胞中,1μmol/L的AngⅡ或100μg/L MFG-E8能够促进AngⅡmRNA的表达(1.62±0.30、2.25±0.36比1.00±0.00,t=3.606、6.015,P<0.05),差异有统计学意义。0.1μmol/L或1.0μmol/L的AngⅡ(1.27±0.11、1.58±0.14比1.00±0.00,t=4.320、6.887,P<0.05),以及50.0μg/L或100.0μg/L的MFG-E8能够促进MFG-E8的表达(1.90±0.09、2.77±0.07比1.00±0.00,t=17.240、43.130,P<0.05),差异有统计学意义。通过AngⅡ刺激HUVECs细胞建立血管高压模型,结果显示,AngⅡ能够显著促进HUVECs内AngⅡ、MFG-E8以及TGF-β1 mRNA的表达(1.62±0.13、4.27±0.47、1.56±0.11比1.00±0.00,t=7.911、12.060、8.710,P<0.01),而AngⅡ受体拮抗剂洛沙钽能抑制AngⅡ的作用,AngⅡ与MFG-E8共处理,则能进一步促进AngⅡ的作用(1.86±0.21比1.00±0.00,t=7.023,P<0.01),显著协同促进MCP-1和MMP-2 mRNA的表达(1.69±0.25、1.69±0.15比1.00±0.00,t=4.735、7.845,P<0.01),差异均有统计学意义。此外,AngⅡ能够显著促进MFG-E8蛋白水平的表达(0.23±0.01比0.16±0.03,t=3.895,P<0.05),而洛沙钽能抑制AngⅡ对MFG-E8蛋白表达的促进作用,AngⅡ与MFG-E8共处理,则能够进一步促进AngⅡ对MFG-E8(0.22±0.03比0.16±0.03,t=2.493,P<0.05)和TGF-β1蛋白水平表达的促进作用(0.Objective To explore the effect of AngiotensinⅡ(AngⅡ)/milk fat globule epidermal growth factor 8(MFG-E8)signaling on matrix remodeling of human umbilical vein endothelial cells(HUVECs)and underlying molecular mechanism.Methods Real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)was used to screen the working concentration of AngⅡand MFG-E8.The correlation of AngⅡwith MFG-E8 and the effect of AngⅡ/MFG-E8 signaling on monocyte chemotactic protein 1(MCP-1),matrix metalloproteinase-2(MMP-2),and transforming growth factor-β1(TGF-β1)expression were analyzed by RT-qPCR and Western blotting.All data were analyzed by SPSS 17.0.The t test was used for pairwise comparison between multiple groups.Results In HUVECs,1μmol/L AngⅡor 100μg/L MFG-E8 could significantly enhance AngⅡmRNA expression(1.62±0.30,2.25±0.36 vs.1.00±0.00,t=3.606,6.015,P<0.05).0.1μmol/L or 1.0μmol/L AngⅡ(1.27±0.11,1.58±0.14 vs.1.00±0.00,t=4.320,6.887,P<0.05),and 50.0 or 100.0μg/L MFG-E8 could significantly increase the expression of MFG-E8 mRNA(1.90±0.09,2.77±0.07 vs.1.00±0.00,t=17.240,43.130,P<0.05).AngⅡstimulation was used to establish a vascular hypertension model in HUVECs,and the data showed that AngⅡsignificantly up-regulated the mRNA expression of AngⅡ,MFG-E8 and TGF-β1(1.62±0.13,4.27±0.47,1.56±0.11 vs.1.00±0.00,t=7.911,12.060,8.710,P<0.01),while co-treatment of AngⅡwith AngⅡreceptor antagonist losartan potassium could inhibit this phenotype.Co-treatment of AngⅡand MFG-E8 further promoted the expression of MCP-1 and MMP-2 mRNA(1.69±0.25,1.69±0.15 vs.1.00±0.00,t=4.735,7.845,P<0.01).In addition,AngⅡcould significantly increase the expression of MFG-E8 in protein level(0.23±0.01 vs.0.16±0.03,t=3.895,P<0.05),while co-treatment of AngⅡand losartan potassium inhibited the effect of AngⅡ.Co-treatment of AngⅡand MFG-E8 further promoted the effect of AngⅡ.Conclusion AngⅡcan enhance the expression of MFG-E8,and MFG-E8 can promote the expression of AngⅡin HUVECs.The A

关 键 词：脐静脉内皮细胞 血管紧张素Ⅱ 表皮生长因子8 

分 类 号：R543.5[医药卫生—心血管疾病]