慢性肾功能衰竭血清对兔主动脉内皮细胞增殖、凋亡及核因子κB信号通路活化的作用  

Potential effect of uremic medium on cell proliferation and apoptosis and the activation of nuclear factor-kappa B pathway of aortic endothelial cells in rabbit culture

作　　者：张耀全[1] 冯兵[2] 胡宏[1] 袁发焕[2] 

机构地区：[1]东南大学医学院附属江阴医院肾内科,214400 [2]第三军医大学新桥医院肾内科全军肾脏病中心

出　　处：《中华医学杂志》2010年第32期2275-2281,共7页National Medical Journal of China

基　　金：基金项目：国家自然科学基金（30570763）

摘　　要：目的 探讨慢性肾功能衰竭（CRF）血清对兔主动脉内皮细胞（AEC）增殖、凋亡和核因子κB信号通路活化及其下游炎症因子表达的影响.方法 建立CRF兔模型,采集其血清 不同浓度的CRF血清刺激原代培养的兔AEC后,四甲基偶氮噻唑盐检测细胞增殖,Hoechst33342染色观察细胞凋亡,流式细胞仪检测细胞周期 反转录-聚合酶链反应（RT-PCR）和Western印迹法分别检测核因子κB、IκB mRNA及蛋白和肿瘤坏死因子（TNF）α蛋白表达 免疫荧光观察核因子κB P65核转位、电泳迁移率变异试验（EMSA）检测核因子κB DNA结合活性 采用酶联免疫吸附试验（ELISA）检测细胞培养液中TNF-α和白细胞介素（IL）6浓度.结果 （1）在较低浓度（≤10%）时,CRF血清对AEC的增殖有明显的促进作用,但血清浓度继续增加后,对AEC的增殖作用却明显减弱,促凋亡作用却增强,与对照组相比,差异均有统计学意义（均P＜0.01）.（2）10%CRF血清刺激后,与对照组相比,核因子κB mRNA（0.35：±0.05比0.26±0.02）及蛋白（1.67±0.15比0.41±0.05）表达均高 IκB mRNA（0.13±0.03比0.24±0.04）和蛋白（0.29±0.06比0.65±0.08）表达均低 核因子κB P65发生核转位 核因子κB DNA结合活性高（均P＜0.01）.与对照组相比,10%CRF血清刺激后AEC分泌TNF-α和IL-6高,细胞中TNF-α蛋白表达高（0.37±0.04比0.14±0.03）（均P＜0.01）.结论 CRF血清可导致AEC活化,低浓度导致AEC增殖,高浓度导致细胞凋亡 促增殖作用可能与其活化了核因子κB信号通路有关.这为进一步采取措施,防治CRF加速性动脉粥样硬化提供了一定的理论和实验依据.Objective To investigate the potential effect of uremic medium on cell proliferation and apoptosis of aortic endothelial cell （AEC）, two key processes in the development of atherosclerosis, in rabbit culture. And to understand the effects of uremic medium on the activation of nuclear factor-kappa B （NF-κB） pathway and cytokines expression of AEC. Methods Rabbit AEC were cultured with growth media supplemented with pooled sera from normal rabbits or those with chronic renal failure. The 80%confiuent AEC were incubated for 24 h with media supplemented with pools of control or uremic sera. Cell proliferation was assessed by a MTT assay and cell cycle detected by flow cytometry. Hoechst33342 assay and flow cytometry were used to investigate the apoptotic effect of uremic medium in AEC. The expression of mRNA and protein levels for NF-κB, IκBα were analyzed by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot. NF-κB P65 nuclear translocation was analyzed by immunofluorescence. The activity of NF-κB was measured by electrophoretic mobility shift assay （EMSA）. Concentrations of TNF-α and IL-6 in culture supernatants were evaluated by ELISA, and the expression of protein for TNF-α in cell lysates by Western blot. Results Uremic medium induced proliferation in the lower concentration range of 3%-10% while promoted apotosis in the higher concentrations （〉10%）. Uremic serum increased NF-κB mRNA （0.35 ±0.05 vs 0.26 ±0.02, P〈0.01） and protein （1.67 ±0.15 vs 0.41 ±0.05, P〈0.01）expression, decreased IκBα mRNA （0.13 ±0.03vs 0.24 ±0.04, P〈0.01） and protein （0.29 ±0.06 vs 0.65 ±0.08, P〈0.01 ） expression. Uremic serum enabled NF-κB p65 nuclear translocation and increased NF-κB DNA binding activity. An increased secretion of cytokines IL-6 and TNF-α. in AEC was observed after a treatment of 10% uremic sera in a time dependent manner. The expression of TNF-α in AEC exposed to 10% uremic sera also increased significantly （0.37 ± 0.04 vs 0.1

关 键 词：肾功能衰竭 慢性 血清 内皮细胞 细胞增殖 NF-κB 

分 类 号：R285.5[医药卫生—中药学]