氧化型低密度脂蛋白对人脐静脉内皮细胞Fractalkine表达的影响及机制分析

目的 观察氧化型低密度脂蛋白对人脐静脉内皮细胞Fractalkine表达的影响并探讨其可能作用机制.方法 采用胰蛋白酶消化法原代培养人脐静脉内皮细胞,取2～5代用于实验,随机分为:①对照组用RPMI1640培养基;②氧化型低密度脂蛋白不同浓度(5、25、50和75 mg/L)组;③氧化型低密度脂蛋白作用不同时间(6、12、24、48和72 h)组;④氧化型低密度脂蛋白+p38MAPK特异性阻断剂SB203580组;⑤p38MAPK特异性阻断剂SB203580组.用RT-PCR及EusA法检测Fractalkine mRNA及蛋白表达水平,用Western blot法检测p38MAPK磷酸化表达水平.结果 ①氧化型低密度脂蛋白在一定浓度范围及作用时间呈时间-剂量依赖方式诱导Fractalkine mRNA及蛋白表达增加,最佳作用时间为48 h,最佳作用浓度为50 mg/L;②与对照组比较,氧化型低密度脂蛋白诱导组人脐静脉内皮细胞p38MAPK磷酸化表达水平显著增加(P<0.05);使用SB203580干预后p38MAPK磷酸化水平明显下降,与氧化型低密度脂蛋白诱导组相比差异有统计学意义(P<0.05).③预先用p38MAPK特异性阻断剂SB203580(20 μmol/L)与内皮细胞共同孵育60 min后,再加入氧化型低密度脂蛋白作用48 h后,Fractalkine表达水平明显降低,与氧化型低密度脂蛋白组比较有统计学差异(P<0.05).结论 氧化型低密度脂蛋白可诱导内皮细胞Fractalkine表达增加,其作用机制可能是通过p38MAPK信号转导通路.

The Effect of ox-LDL on the Expression of Fractalkine in Cultured Human Umbilical Vein Endothelial Cells and Its Mechanism

Aim To investigate the effect of oxidized low density lipoprotein(ox-LDL) on Fraetalkine expression in cultured human umbilical vein endothelial cells (HUVEC) and explore its mechanism.Methods HUVECs were isolated and cultured, the passage 2 ～ 5 cells were used in experiment. HUVEC were coincubated with different concentrations of ox-LDL (5, 25, 50 and 75 mg/L) for 48 h or with 50 mg/L ox-LDL for different times (6, 12, 24, 48 and 72 h). SB203580(20 μmol/L), an inhibitor of p38MAPK activation, was pretreated for 60min before HUVEC were coincubated with 50 mg/L ox-LDL for 48 h. Fractalkine mRNA and protein expression were detected by RT-PCR or ELISA. The phosphorylation of p38MAPK was determined by Western blot.Results The expressions of fraetalkine in HUVEC were upregulated by ox-LDL(5, 25 and 50 mg/L) in a concentration- dependent manner and were increased by ox-LDL(50 mg/L) for6, 12, 24, 48 and 72 h in a time-dependent manner; Compared with control group, the phosphorylation of p38MAPK was increased significantly in ox-LDL group (P < 0.05), SB203580 (20 μmol/L) decreased ox-LDL induced fractalkine expression.Conclusion ox-LDL stimulates the expression of fractalkine in a concentration and time dependent manner, p38MAPK activation may mediate ox-LDL induced fractalkine expression.