摘要目的 探讨多药耐药相关蛋白4 (MRP4)对脂多糖(LPS)诱导的大鼠肺微血管内皮细胞(PMVECs)通透性及细胞骨架的影响.方法 PMVECs细胞株培养3至6代后随机分为4组,对照组(细胞不做任何处理)、LPS组(细胞仅接受LPS刺激)、shMRP4重组腺病毒组(Ad-shMRP4,将针对大鼠MRP4基因设计的短发夹RNA构建腺病毒载体上,通过腺病毒感染细胞+LPS刺激)、shRNA重组腺病毒组(Ad-shRNA,带绿色荧光蛋白基因空白的重组腺病毒载体感染细胞+ LPS刺激).通过荧光显微镜观察细胞感染率,Western botting检测MRP4的表达水平,Transwell小室法检测单层细胞通透性,激光共聚焦显微镜观察细胞内纤维肌动蛋白(F-actin)形态及分布情况.多组间差异采用单因素方差分析,组间两两比较采用LSD-t检验,以P＜0.05为差异有统计学意义.结果 与对照组比较,LPS组及Ad-shRNA组MRP4的表达水平显著上调(P＜0.05)、单层细胞通透性显著增加(2、6、12、24h分别为:0.28±0.02和0.41±0.04、0.32±0.02;0.30±0.01和0.53±0.04、0.39±0.03;0.33±0.04和1.12±0.17、0.70±0.07;0.32±0.03和0.79±0.02、0.57±0.05,P＜0.05),F-actin分布紊乱、重构以及应力纤维形成.与LPS组比较,Ad-shMRP4组MRP4的表达水平明显下调(P＜0.05)、单层细胞通透性明显降低(2、6、12、24 h分别为:0.41±0.04和0.32±0.02;0.53±0.04和0.39±0.03;1.12±0.17和0.70±0.07;0.79±0.02和0.57±0.05,P＜0.05),F-actin重构以及应力纤维形成显著减少.结论 MRP4基因沉默显著增减轻LPS诱导的内皮细胞通透性增高及细胞骨架破坏,对内皮细胞屏障发挥保护作用.

abstractsObjective To investigate the effects of multidrug resistance-associated protein 4 (MRP4) on the cytoskeleton and cellular permeability of rat pulmonary microvascular endothelial cells (PMVECs) induced by lipopolysaccharide (LPS).Methods PMVECs were cultured for 3 to 6 generations were randomly divided into 4 groups:control group,LPS group,Ad-shMRP4 group (adenoviral expression of a short-hairpin RNA directed against MRP4),Ad-shRNA group.The infection rate of cells was detected by fluorescence microscope observation.The level of MRP4 was assayed by Western botting.Monolayer permeability was determined by the Transwell assay.The morphological characteristic and distribution of Factin was measured by laser confocal fluorescence microscope.Results Compared with control group,the expression of MRP4 protein was up-regulated (P ＜ 0.05) and the significant increase in the permeability of endothelial cells (2 h,6 h,12 h and 24 h respectively:0.28 ±0.02 vs.0.41 ±0.04,0.32 ±0.02,0.30 ±0.01 vs.0.53±0.04,0.39±0.03,0.33 ±0.04 vs.1.12±0.17,0.70 ±0.07,0.32±0.03 vs.0.79 ± 0.02,0.57 ± 0.05,P ＜ 0.05),the F-actin was remodeled,and the stress fibers were formed in LPS group and Ad-shMRP4 group.However,compared with LPS group,the expression of MRP4 protein was down-regulated (P ＜ 0.05) and the markedly decrease in the permeability of endothelial ceils (2 h,6 h,12 h and 24 h respectively:0.41 ± 0.04 vs.0.32 ± 0.02,0.53 ± 0.04 vs.0.39 ± 0.03,1.12 ± 0.17 vs.0.70 ± 0.07,0.79 ± 0.02 vs.0.57 ± 0.05,P ＜ 0.05) was found,and the remodeling of F-actin,and the formation of stress fibers were observed in Ad-shMRP4 group.Conclusions Silencing of MRP4 gene can effectively attenuates LPS-induced increase in the endothelial cell permeability and the destruction of cytoskeleton,thus playing an important role in the protection of endothelial cell barrier.