RNA干扰人血凝素样氧化型低密度脂蛋白受体表达对内皮细胞骨架损伤的影响

目的构建针对人血凝素样氧化型低密度脂蛋白受体1(LOX-1)基因的RNA干扰慢病毒载体,并转染人脐静脉内皮细胞后,采取氧化型低密度脂蛋白(ox-LDL)诱导观察对内皮细胞骨架损伤的影响。方法体外培养人脐静脉内皮细胞分为对照组、ox-LDL组(ox-LDL处理)、阴性转染组(ox-LDL处理+阴性慢病毒转染)和慢病毒转染组(ox-LDL处理+最佳干扰序列慢病毒转染)。荧光显微镜观察转染效率,Western blot检测磷酸化肌球蛋白轻链(p-MLC)、Rho激酶(ROCK)、LOX-1蛋白的表达;免疫荧光法观察细胞骨架肌动蛋白(F-actin)的变化。结果与对照组比较,ox-LDL组和阴性转染组p-MLC、ROCK、LOX-1蛋白表达增高;细胞F-actin发生损伤并伴含量减少(P<0.05)。与阴性转染组比较,慢病毒转染组抑制p-MLC、ROCK、LOX-1蛋白表达,减轻F-actin损伤及伴含量增加(P<0.05)。结论干扰LOX-1表达对ox-LDL诱导引起的内皮细胞ROCK、p-MLC表达增加及细胞骨架损伤均有抑制作用。

Effect of RNA-intefered lectin-like LOX-1 expression on oxidized LDL-induced injury of endothelial cell cytoskeleton

Objective To study the effect of RNA-interfered lentiviral vector which was transfected into human umbilical vein endothelial cells(HUVEC)on oxidized LDL-induced injury of endothelial cell cytoskeleton.Methods In vitro-cultured HUVEC were divided into normal control group,oxidized LDL treatment group,negative transfection group(oxidized LDL+negative lentiviral transfection),lentivirial transfection group(oxidized LDL+optimal lentiviral transfection interference).The transfection efficiency was observed by fluorescence microscopy.Expressions of p-MLC,Rho kinase and LOX-1were detected by Western blot.Changes of F-actin in endothelial cytoskeletal protein were observed using the immunofluorescence technique.Results The expression levels of p-MLC,Rho kinase and LOX-1were significantly higher while the number and level of injured F-actin were significantly lower in oxidized LDL treatment group and negative transfection group than in normal control group(P<0.05).The expression levels of p-MLC,Rho kinase and LOX-1were significantly lower while the number and level of injured F-actin were significantly higher in lentivirial transfection group than in negative transfection group(P<0.05).Conclusion Interference of LOX-1expression increases the expression of Rho kinase and p-MLC protein and decreases oxidized LDL-induced injury of endothelial cell cytoskeleton.