摘要
目的研究脂多糖(LPS)作用下血管内皮钙黏蛋白(VE-Cad)经不同的胞吞途径进入细胞后对VE-Cad亚细胞分布和细胞通透性的影响。方法体外培养人血管内皮细胞株CRL-2922,观察LPS(10μg/ml)作用后不同时间点VECad与R ab11(循环内颗粒标记物)及溶酶体相关膜蛋白2(L AMP2,晚期内颗粒/溶酶体标记物)的免疫共沉淀情况,网格蛋白胞吞抑制剂和微囊抑制剂对VE-Cad与Rab11和LAMP2免疫共沉淀的影响,以及单层细胞通透性的变化。结果LPS作用后1h,VE-Cad与Rab11的免疫共沉淀明显增高(P<0.05),随后逐渐降低;VE-Cad与LAMP2的免疫共沉淀在LPS作用后呈时间依赖性地增高(P<0.05)。网格蛋白胞吞抑制剂氯丙嗪(CPZ)可显著抑制LPS作用后VE-Cad与Rab11免疫共沉淀的增高(P<0.05),而微囊抑制剂非律平无此作用;微囊抑制剂非律平可显著抑制LPS作用后VE-Cad与LAMP2免疫共沉淀的增高(P<0.05),而网格蛋白胞吞抑制剂无此作用。网格蛋白胞吞抑制剂可减轻LPS作用后1h单层细胞通透性的增高,而微囊抑制剂可减轻LPS作用后4h单层细胞通透性的增高(P<0.05)。结论在LPS作用下VE-Cad分别经网格蛋白或微囊介导的胞吞进入细胞,分布于循环内颗粒或溶酶体中,从而导致不同程度的血管通透性增高。
Objective To investigate different subcellular distribution and the effect on cell permeability of vascular endothelial cadherin(VE-Cad) in cytoplasm via clathrin- and caveolae-mediated endocytosis pathways after lipopolysaccharide(LPS) challenge. Methods The co-immunoprecipitation of VE-Cad w ith R ab11(marker of the rec ycling endosomes) and lysosome-associated membrane protein 2(L AMP2, marker of the late endosomes/lysosomes) at dif ferent time points, as well as the effects of clathrin-mediated endocy tosis inhibitor chlorpromazine(CPZ) and caveolae inhibitor filipin on the coimmunoprecipitation of VE-Cad with Rab11/LAMP2 and on the monolayer cell permeability after LPS(10μg/ml) treatment, were obser ved. Results 1) The co-immunoprecipitation of VE-Cad with Rab11 was increased at 1h after LPS treatment(P〈0.05), and then it decreased gradually, while the co-immunoprecipitation of VE-Cad with L AMP2 was increased with elapse of time after LPS treatment(P〈0.05). 2) The increased co-immunoprecipitation of VE-Cad with Rab11 was inhibited obviously by the clathrin-mediated endocytosis inhibitor CPZ(P〈0.05), while the inhibitor of caveolae filipin failed to do so. The increased coimmunoprecipitation of VE-Cad w ith L AMP2 was inhibited obv iously by the caveolae filipin(P〈0.05), while the clathrinmediated endocytosis inhibitor CPZ failed. 3) The increased monolayer cell permeability at 1h after LPS treatment was improved by the clathrin-mediated endocytosis inhibitor CPZ(P〈0.05), and the increased monolayer cell hyperpermeability at 4h after LPS treatment could be significantly attenuated by the caveolae inhibitor filipin. Conclusion VE-Cad may get into the cells and locate in recycling endosomes or lysosomes respectively through clathrin- and caveolae-mediated endocytosis after LPS treatment, thus leading to hyperpermeability in different extent of monolayer cells.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2016年第1期22-25,共4页
Medical Journal of Chinese People's Liberation Army
基金
“十二五”国家科技支撑计划(2012BAI11B01)
军队“十二五”医学科研重点项目(BWS12J033)