钙库操纵性钙内流参与调节ox-LDL诱导的血管内皮细胞通透性改变  

Store-operated calcium entry involves in regulating the hyperpermeability of endothelial cells induced by ox-LDL

作　　者：康越 邰苏豫 李茂巍 于林君 王连友 KANG Yue;TAI Suyu;LI Maowei;YU Linjun;WANG Lianyou(School of Graduate Students,Xinxiang Medical University,Xinxiang,Henan Province,453003;Department of Cardiology,No.988 Hospital of Joint Logistic Force of PLA,Zhengzhou,Henan Province,450042,China)

机构地区：[1]新乡医学院研究生院,河南新乡453003 [2]联勤保障部队988医院心内科,郑州450042

出　　处：《陆军军医大学学报》2023年第4期358-365,共8页Journal of Army Medical University

基　　金：河南省医学科技攻关计划(联合共建)项目(LHGJ20190865)。

摘　　要：目的利用体外培养的人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs),探讨钙库操纵性钙内流(store-operated calcium entry,SOCE)在氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)诱导的血管内皮细胞通透性改变中的作用。方法体外培养HUVECs,传代3次后,用于检测跨内皮电阻(transendothelial electrical resistance,TER)的细胞随机分为对照组和不同剂量ox-LDL(10、25、50、100μg/mL)干预组(n=3)。培养于载玻片上的细胞随机分为对照组和ox-LDL 24 h组、ox-LDL 48 h组,免疫荧光检测细胞骨架蛋白F-actin及细胞连接蛋白VE-cadherin的分布,免疫印迹检测VE-cadherin的含量。细胞孵育钙离子探针(Fluo-3/AM)后,激光共聚焦检测细胞内钙含量(对照组、ox-LDL组、ox-LDL+2APB组)以及SOCE(对照组、ox-LDL 24h组、ox-LDL 48 h组)。结果低剂量的ox-LDL(10、25μg/mL)对TER无明显影响,高剂量ox-LDL(50、100μg/mL)在6 h内对TER影响不明显,但在12 h引起TER降低(P<0.05),此效应在24 h和48 h更加明显(P<0.05)。Ox-LDL(100μg/mL)处理后24、48 h,VE-cadherin的表达减少(P<0.05),并影响F-actin的形态和分布;HUVECs的游离钙浓度显著增加(P<0.05),此效应被SOCE抑制剂2-APB阻断,2-APB能够减弱ox-LDL对TER的影响。ox-LDL(100μg/mL)处理48 h后,SOCE的振幅显著降低(P<0.05)。SOCE降支速度在ox-LDL处理24 h后呈现降低趋势,而在ox-LDL处理48 h后呈显著下降(P<0.05)。结论ox-LDL通过延长内皮细胞SOCE的持续时间上调细胞内游离钙浓度,影响细胞骨架的排列和细胞间连接的完整性,引起细胞通透性的增加。Objective To explore the effects of store-operated calcium entry(SOCE)on oxidized low-density lipoprotein(ox-LDL)-induced hyperpermeability in human umbilical vein endothelial cells(HUVECs).Methods HUVECs at passage 3 were divided into the control group and the ox-LDL(10,25,50 and 100μg/mL)intervention groups(n=3)for the examination of transendothelial electrical resistance(TER).HUVECs cultured in slides were randomly divided into the control group,the ox-LDL 24 h group and the ox-LDL 48 h group.The distribution of F-actin and VE-cadherin were detected by immunofluorescence,and the protein content of VE-cadherin was detected by Western blotting.Incubated with calcium fluorescence probe(Fluo-3/AM),intracellular free Ca2+concentration(control group,ox-LDLgroup,ox-LDL+2-APB group)and SOCE(control group,ox-LDL 24 h group,ox-LDL 48 h group)were detected by laser confocal microscopy.Results Low dose of ox-LDL(10,25μg/mL)exerted no effect on TER.High dose of ox-LDL(50,100μg/mL)also had no effect on TER within 6 h,but after 6 h showed no effect on TER.However,high dose of ox-LDL(50,100μg/mL)reduced the decrease of TER in 12 h(P<0.05),and the decrease was more significant in 24 h and 48 h.After the incubation with ox-LDL(100μg/mL)24 h and 48 h,the expression level of VE-cadherin decreased(P<0.05),while the form and distribution of F-action changed;Intracellular free Ca2+concentration increased obviously which was interrupted by 2-APB the SOCE inhibitor(P<0.05),and the 2-APB can decrease the effect of ox-LDL on TER.After incubation with ox-LDL(100μg/mL)48 h,the amplitude of SOCE decreased(P<0.05).The descending artery flow velocity of SOCE showed a slightly decreased trend after incubation with ox-LDL 24 h and a more significant decrease in 48 h(P<0.05).Conclusion Ox-LDL up-regulate the intracellular free Ca2+concentration of vascular endothelial cells by extending the duration of SOCE to effect the arrangement of cytoskeleton and cell junction,which increases the cell permeability.

关 键 词：钙库操纵性钙内流 细胞骨架 氧化-低密度脂蛋白 通透性 血管内皮细胞 

分 类 号：R322.123[医药卫生—人体解剖和组织胚胎学] R329.27[医药卫生—基础医学] R363.21