网格蛋白介导胞吞在血管通透性增高中的作用  

Contributions of clathrin-mediated endocytosis of vascular endothelial cadherin to lipopolysaccharide-induced vascular hyperpermeability

作　　者：张晔[1] 张连阳[1] 李阳[1] 谭浩[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：《中华实验外科杂志》2015年第11期2681-2684,共4页Chinese Journal of Experimental Surgery

基　　金：“十二五”国家科技支撑计划资助项目（2012BAI11B01）;军队“十二五”重点资助项目（BWS12J033）

摘　　要：目的 观察网格蛋白介导的血管内皮钙黏蛋白（VE-Cad）胞吞在脂多糖（LPS）诱导血管通透性增高中的作用.方法 以人血管内皮细胞株CRL-2922为研究对象,采用Western blot、免疫共沉淀、免疫组织化学等方法,观察LPS处理不同时间点的VE-Cad蛋白表达和单层细胞通透性、网格蛋白与VE-Cad的共沉淀和共定位,以及网格蛋白胞吞抑制剂对LPS处理后网格蛋白与VE-Cad的共沉淀、VE-Cad蛋白表达和单层细胞通透性的影响.结果 （1）LPS（10 mg/L）处理后VE-Cad的质膜表达逐渐降低（0.621 ±0.092降至0.162±0.035,P＜0.05）,单层细胞通透性逐渐增高（0.263±0.042增至0.751 ±0.102,P＜0.05）;（2） LPS处理后网格蛋白表达逐渐降低（0.525±0.047降至0.270±0.032,P＜0.05）,与VE-Cad的免疫共沉淀在1h时增高（0.289±0.055,P＜0.05）,之后逐渐降低,免疫组织化学激光共聚焦显微镜观察其共定位显示同样的变化;（3）网格蛋白胞吞抑制剂氯丙嗪（CPZ,100μmol/L）可以显著降低LPS处理1h网格蛋白与VE-Cad的免疫共沉淀（0.127±0.034,P＜0.05）,增高VE-Cad的质膜表达（0.603±0.071,P＜0.05）,改善单层细胞通透性（0.319±0.045,p＜0.05）;但对LPS处理4h网格蛋白与VE-Cad的免疫共沉淀、VE-Cad质膜表达以及单层细胞通透性没有显著影响.结论 网格蛋白介导VE-Cad胞吞参与LPS作用早期血管通透性增高的形成,但不影响LPS作用后期的血管通透性增高.Objective To observe the role of clathrin-mediated endocytosis of vascular endothelial cadherin （VE-cad） to lipopolysaccharide （LPS）-induced vascular hyperpermeability.Methods Human vascular endothelial cell line CRL-2922 was adapted as the research subject, Western blotting, co-immunoprecipitation, and immunocytochemistry were adapted to observe the protein expression of VE-cad and monolayer cell permeability after LPS treatment, clathrin-mediated endocytosis of VE-cad and effect of clathrin-mediated endocytosis inhibitor on the clathrin-mediated endocytosis of VE-cad, the protein expression of VE-cad and monolayer cell permeability.Results （1） Normal cells displayed strong VE-cad expression in plasma membrane, and an decreased plasma membrane VE-cad was found after LPS （10 mg,/L） treatment （from 0.621 ±0.092 to 0.162 ±0.035, P 〈0.05）;the monolayer cell permeability was increased gradually in a time-dependent manner after LPS treatment （from 0.263 ± 0.042 to 0.751 ±0.102, P 〈0.05）.The monolayer cell permeability and the VE-cad location in plasma membrane were negatively correlated.（2） The protein expression of clathrin was decreased gradually after LPS treatment （from 0.525 ± 0.047 to 0.270 ± 0.032, P 〈 0.05）;the co-immunoprecipitation of V E-cad with clathrin was low in normal control, increased at 1 h after LPS treatment （0.289 ± 0.055, P 〈 0.05）, and decreased as time went on;the co-localization of VE-cad and clathrin showed the accordant change with that of co-immunoprecipitation.（3） The clathrin-mediated endocytosis inhibitor CPZ （100 μmol/L） could decrease the co-immunoprecipitation of VE-cad and clathrin （0.127 ± 0.034, P 〈 0.05）, increase the VE-cad location in plasma membrane （0.603 ± 0.071, P 〈 0.05）, and improve the monolayer cell permeability （0.319 ±0.045, P 〈0.05） at 1 h after LPS treatment, despite of no change at 4 h after LPS treatment.Conclusion Clathrin-mediated endocytosis of VE-cad led to the decrease of VE-cad

关 键 词：血管通透性增高 脂多糖 血管内皮钙黏蛋白 网格蛋白 

分 类 号：R363[医药卫生—病理学]