脂多糖通过TLR4-Src信号介导微囊胞吞血管内皮钙黏蛋白和增加血管通透性  

Contribution of TLR4-Src Signal Pathway to Caveolae-mediated Endocytosis of VE-Cad after LPS Treatment

作　　者：张晔[1] 张连阳[1] 谭浩[1] 李阳[1] 孙士锦[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所创伤中心,创伤,烧伤与复合伤国家重点实验室,重庆400042

出　　处：《微循环学杂志》2015年第2期1-4,共4页Chinese Journal of Microcirculation

基　　金："十二五"国家科技支撑计划(2012BAI11B01);军队"十二五"重点项目(BWS12J033)

摘　　要：目的:探讨脂多糖(LPS)诱导微囊胞吞血管内皮钙黏蛋白(VE-Cad)的可能机制。方法:培养人血管内皮细胞株CRL-2922,当其生长至融合状态时分为正常对照组(不予再处理)、LPS处理组(采用10μg/ml LPS分别与CRL-2922再培养1h、2h、4h和6h)、LPS+抑制剂组包括Toll样受体4(TLR4)抑制剂CLI-095和Src抑制剂SU6566[在LPS培养细胞时分别加入CLI-095(5μg/ml)和SU6566(2μmol/L)再培养4h]。采用Western Blotting法检测各组Src蛋白表达、Cav1磷酸化和VE-Cad质膜蛋白表达,以及Cav1与VE-Cad共沉淀水平;采用培养小室半透膜培养细胞,并检测相关各组细胞荧光透过率,以反映血管通透性。结果:LPS处理不同时间组Src蛋白表达均较正常对照组升高(P<0.05);与正常对照组比较,LPS处理4h组Cav1磷酸化增强(P<0.05)、VE-Cad质膜蛋白表达下调、Cav1与VE-Cad共沉淀水平升高(P<0.05),单层细胞荧光透光率增加(P<0.05);TLR4抑制剂和Src抑制剂可显著降低LPS增高的Src蛋白高表达和Cav1高度磷酸化(P<0.05),上调VE-Cad质膜蛋白表达(P<0.05),下调Cav1与VE-Cad共沉淀水平(P<0.05),改善单层内皮细胞通透性(P<0.05)。结论:LPS可能通过TLR4-Src信号途径诱导微囊胞吞VE-Cad和增加血管通透性。Objective：To observe the mechanism of caveolae-mediated endocytosis of VE-Cad after LPS treat-ment.Method：Human vascular endothelial cell line CRL-2922 was cultured.It was divided into normal control group （when it grew to confluence state）,LPS-treated group （using 10μg/ml LPS to incubate with the CRL-2922 for 1h,2h,4h and 6h）;and LPS＋inhibitor group[adding CLI-095 （5μg/ml）and SU6566 （2μmol/L）in the LPS cultured cells and then culturing for 4h].Western Blotting was used to detect the Src protein expression,Cav1 phosphorylation,plasma membrane protein expression of VE-Cad,phosphorylation of Cav1 and co-precipitation lev-els of Cav1 and VE-Cad;semi-permeable membrane was used to culture cells,and the relevant cells fluorescence transmittance was detected to reflect vascular permeability.Results：The protein expression of Src was gradually in-creased after LPS treatment,as well as the phosphorylation （Tyr14）of Cav1 and he monolayer cell permeability （P〈0.05）,and the membrane expression of VE-Cad decreased （P〈0.05）.The increased expression and phosphorylation could be decreased by the inhibitor of TLR4 CLI-095 and the inhibitor of Src SU6656 （P〈0.05）,and the inhibitors could increase the membrane expression of VE-Cad （P〈0.05）,and improve the monolayer cell permeability at 4h after LPS treatment （P〈0.05）.Conclusion：Caveolae-mediated endocytosis of VE-Cad was activated through LPS-TLR4-Src signal pathway.

关 键 词：脂多糖 Toll样受体4 Src蛋白 微囊胞吞血管内皮钙黏蛋白 血管通透性 

分 类 号：R605.971[医药卫生—急诊医学]