细胞骨架改构在LPS作用后VE-Cad胞吞途径转化中的作用

Role of Cytoskeleton Structure in The Transformation of Endocytosis Pathways of Vascular Endothelial Cadherin after Lipopolysaccharide Treatment

目的探索细胞骨架解聚剂细胞松弛素D(Cyt D)和稳定剂Jasplakinolide(Jasp)通过改构细胞骨架结构对LPS作用后网格蛋白/微囊介导的血管内皮细胞钙黏蛋白(VE-Cad)胞吞、膜VE-Cad表达和血管通透性的影响。方法采用CRL-2922细胞进行实验,各组均于组别对应的时点检测(空白对照组任意时点皆可)。1将细胞分为空白对照组、LPS-1 h组及LPS-4 h组,观察细胞骨架。2将细胞分为LPS-1 h组、Cyt D+LPS-1 h组、LPS-4 h组及Jasp+LPS-4 h组,检测网格蛋白/Cav1与VE-Cad共沉淀和膜VE-Cad的表达水平。3将细胞分为空白对照组、LPS-1 h组、Cyt D+LPS-1 h组、LPS-4 h组及Jasp+LPS-4 h组,检测单层细胞的累积荧光透过率。结果 1空白对照组中肌动蛋白呈均匀散在分布,细胞骨架无明显的聚合;LPS-1 h组中细胞骨架发生明显的聚合,张力丝形成;LPS-4 h组中细胞骨架解聚,张力丝消失。2与LPS-1 h组比较,Cyt D+LPS-1 h组中网格蛋白与VE-Cad的共沉淀水平较低(P<0.05),Cav1与VE-Cad的共沉淀水平较高(P<0.05),且膜VE-Cad的表达水平降低(P<0.05);与LPS-4 h组比较,Jasp+LPS-4 h组中网格蛋白与VE-Cad的共沉淀水平的差异无统计学意义(P>0.05),Cav1与VE-Cad的共沉淀水平较低(P<0.05),且膜VE-Cad的表达水平升高(P<0.05)。3与空白对照组比较,LPS-1 h组和LPS-4 h组的累积荧光透过率均较高(P<0.05);与LPS-1 h组比较,Cyt D+LPS-1 h组的累积荧光透过率较高(P<0.05);与LPS-4 h组比较,Jasp+LPS-4 h组的累积荧光透过率较低(P<0.05)。结论 LPS作用后细胞骨架先发生聚合然后解聚,这种改构促使VE-Cad胞吞途径从由网格蛋白介导转化到由微囊介导。

Objective To explore the effects of cytoskeleton depolymerizing agent and stabilizer on the clathrin/caveolae-mediated endocytosis, the expression of membrane vascular endothelial cadherin（VE-cad）, and the vascular permeability by the transformation of cytoskeleton structure after lipopolysaccharide（LPS） treatment. Methods CRL-2922 cells were used in the experiments. Indexes were tested at corresponding time point according to name of group, but in blank control group indexes could be tested at any time point. CRL-2922 cells were divided into blank control group, LPS-1 h group, and LPS-4 h group to observe cytoskeleton structure; CRL-2922 cells were divided into LPS-1 h group,Cyt D＋LPS-1 h group, LPS-4 h group, and Jasp＋LPS-4 h group to determine the expression of membrane VE-cad,and to determine the expression of its co-immunoprecipitation with clathrin and caveolin-1（Cav1）; besides, CRL-2922 cells were divided into blank control group, LPS-1 h group, Cyt D＋LPS-1 h group, LPS-4 h group, and Jasp＋LPS-4 h group to detect the cumulative infiltration rate. Results 1 The cytoskeleton showed a dynamic change after LPS treatment, the F-actin polymerized and stress fibers formed at 1 hour after LPS treatment, but depolymerized at 4 hours after LPS treatment. 2 Compared with LPS-1 h group, the level of co-immunoprecipitation of VE-cad with clathrin in Cyt D＋LPS-1 h group decreased（P〈0.05）, the level of co-immunoprecipitation of VE-cad with Cav1 increased（P〈0.05）, and expression level of VE-cad in plasma membrane decreased（P〈0.05）; compared with LPS-4 h group, there was no significant difference in the level of co-immunoprecipitation of VE-cad with clathrin of Jasp＋LPS-4 h group（P〉0.05）, but the level of co-immunoprecipitation of VE-cad with Cav1 decreased in Jasp＋LPS-4 h group（P〈0.05）, and expression level of VE-cad in plasma membrane increased（P〈0.05）. 3 Compared with blank control group, the cumulative infiltration rates of LPS-1 h group and LPS-4 h group were both higher（P〈0.05）; compared with LPS-1 h group, the cumulative infiltration rate of Cyt D＋LPS-1 h group was higher（P〈0.05）; compared with LPS-4 h group, the cumulative infiltration rate of Jasp＋LPS-4 h group was lower（P〈0.05）. Conclusion Actin cytoskeleton shifts from polymerization to depolymerization after LPS treatment, the structural change of actin cytoskeleton is an important reason for the transformation of VE-cad endocytosis pathway from clathrin-mediated to caveolae-mediated after LPS treatment.