微囊介导 VE－Cad 胞吞参与了 LPS 作用后血管通透性增高的形成

Caveolae-mediated endocytosis of VE-Cad contributes to vascular hyperpermeability after LPS treatment

目的:观察脂多糖(lipopolysaccharide,LPS)作用后微囊介导的血管内皮钙黏蛋白(vascular endothelial cadherin,VE-Cad)胞吞,及其在血管通透性增高中的作用。方法:采用人血管内皮细胞株CRL-2922,以及免疫组化、免疫印迹、免疫共沉淀等技术方法,观察LPS处理后不同时点细胞质膜微囊重要结构蛋白小窝蛋白1(caveolin-1,Cav1)的蛋白表达和磷酸化,Cav1与VE-Cad的共沉淀和共定位,以及微囊抑制剂对LPS处理后Cav1与VE-Cad的共沉淀、VE-Cad质膜表达和单层细胞通透性的影响。结果:(1)LPS处理后Cav1蛋白表达无显著变化,但其Tyr14位点的磷酸化水平逐渐增高(P<0.05),Cav1与VE-Cad的免疫共沉淀逐渐增多(P<0.05),免疫组化激光共聚焦显微镜观察在4 h时可见明显的共定位;(2)细胞质膜微囊抑制剂非律平(5 mg/L)可以显著减少LPS处理4 h Cav1与VE-Cad的免疫共沉淀(P<0.05),增强VE-Cad的质膜表达(P<0.05),改善单层细胞通透性(P<0.05)。结论:细胞质膜微囊介导VE-Cad胞吞参与了LPS作用后血管通透性增高的形成。

AIM： To observe caveolae-mediated endocytosis of vascular endothelial cadherin （VE-Cad） after lipopolysaccharide （LPS） treatment, and its role in vascular hyperpermeability. METHODS： Human vascular endothelial cell line CRL-2922 was used in the experiment. Western blot, co-immunoprecipitation and immunocytochemistry were adopted to observe the protein expression of caveolin-1 （ Cavl ）, a main structural protein of caveolae, after LPS treatment. Caveolae-mediated endocytosis of VE-Cad after LPS treatment, and the effects of caveolae inhibitor on caveolae-mediated endocytosis of VE-Cad, the protein expression of VE-Cad and monolayer cell permeability after LPS treatment were deter- mined. RESULTS ： After LPS treatment, the protein expression of Cavl did not show a significant change, while the phos- phorylation （Tyrl4） of Cavl was significantly increased （ P 〈 0.05 ）. No co-immunoprecipitation or co-localizatian of VE- Cad with Cavl was observed in the normal control, and that was increased time-dependently after LPS treatment （P 〈 0.05）. Filipin, an inhibitor of caveolae, at concentration of 5 mg/L significantly reduced the eo-immunoprecipitation of VE-Cad with Cavl （P 〈0. 05）, increased the expression of VE-Cad （P 〈0. 05） in the membrane, and improved the mon- olayer cell permeability at 4 h after LPS treatment （ P 〈 0. 05 ）. CONCLUSION ： Caveolae-mediated endocytosis of VE- Cad contributes to the internalization of VE-Cad and the monolayer cell hyperpermeability after LPS treatment.