烧伤血清对内皮细胞核因子-κB核移位的影响

目的　了解烧伤血清对内皮细胞核因子 κB (NF κB)异二聚体 p5 0 /p6 5核移位 ,以及核抑制因子κB(IκB)α降解的影响 ,进一步探讨烧伤血清对内皮细胞的活化作用。方法　采用体外培养的人脐静脉内皮细胞株ECV 30 4 ,分别用正常人血清、烧伤患者血清、烧伤患者血清 吡咯烷二硫代氨基甲酸盐 (PDTC)刺激内皮细胞 ,并以正常培养的内皮细胞为对照。应用激光共聚焦显微镜观察刺激 30、6 0、12 0、4 80min后内皮细胞 p5 0 /p6 5核移位情况 ,采用Western印迹法检测刺激 30、6 0、90、12 0min后内皮细胞IκBα蛋白降解的情况。　结果　与对照组比较 ,烧伤血清刺激内皮细胞 30min后 ,p5 0、p6 5即发生核移位 ,30～ 6 0min达高峰 ,2h后恢复至刺激前状态 ;而烧伤血清刺激 30min后 ,IκBα发生降解 (P <0 .0 1) ,刺激 4 5～ 6 0min后最为明显 ,2h后表达逐渐恢复。PDTC能有效抑制烧伤血清作用 30、6 0min后 ,p5 0、p6 5的核移位和IκBα降解。 　结论　烧伤血清可导致内皮细胞NF κBp5 0、p6 5发生核移位 ,并使IκBα降解 ,进而活化NF κB ,诱导内皮细胞分泌细胞因子。PDTC对这一系列变化可能有抑制作用

The influence of burn sera on the nuclear translocation of endothelial NF-κB p50 and p65

OBJECTIVE: To investigate the effects of burn sera on the nuclear translocation of endothelial NF-kappaB heterodimers p50/p65 and on the degradation of inhibiting kappaB (IkappaBalpha), in order to explore the role of burn sera on activation of the endothelium. METHODS: Cultured human umbilical vein endothelial cells (HUVECs) (ECV-304 strain) were employed as the target cells. The cells were stimulated by sera from healthy volunteers and from burn patients and burn sera together with PDTC (pyrrolidine dithiocarbarnate). The normal cultured cells were taken as the control. The nuclear translocation of endothelial p50/p65 at 30, 60, 120 and 480 mins after the stimulation was observed with laser confocal microscopy, and the endothelial IkappaBalpha protein degradation at 30, 60, 90 and 120 mins after the stimulation was determined by Western blotting. RESULTS: When compared to that in control group, the nuclear translocation of p50/p65 took place 30 mins after the endothelial cells were stimulated by burn sera, and it reached the summit at 30 - 60 mins, but recovered to pre-stimulation state at 2hrs. In addition, IkBalpha degradation occurred 30 mins after the cells were stimulated by burn sera (P < 0.01) and peaking at 45 - 60 mins after the stimulation and recovered at 2hrs after the stimulation. The nuclear translocation of endothelial p50/p65 and IkBalpha degradation at 30 and 60 mins after the stimulation by burn sera could be effectively inhibited by PDTC. CONCLUSION: Burn sera might induce the nuclear translocation of endothelial NF-kappaB p50/p65 and IkappaBalpha degradation and activate NF-kappaB, which ultimately lead to the secretion of cytokines from the endothelium.