库存红细胞增强脂多糖诱导的外周血单个核细胞炎症反应机制的研究  

Study on the mechanism of enhanced inflammatory response of peripheral blood mononuclear cells induced by lipopolysaccharide by banked red blood cells

作　　者：王晓伟[1] 任召祺[2] 王海平[1] 卓海龙[1] 张莎娜[1] 曹慧[1] 王全立[1] 

机构地区：[1]军事医学科学院附属医院输血科,北京100071 [2]第二炮兵总医院输血科,北京100088

出　　处：《感染．炎症．修复》2012年第2期109-112,共4页Infection Inflammation Repair

摘　　要：目的:研究库存红细胞对内毒素(LPS)诱导的外周血单个核细胞(PBMC)释放炎性细胞因子的影响及其机制。方法:分离健康献血员的PBMC,分为PBMC组、PBMC+SN50组、PBMC+LPS组、PBMC+LPS+SN50组、PBMC+d1组、PBMC+d1+SN50组、PBMC+d1+LPS组、PBMC+d1+LPS+SN50组、PBMC+d35组、PBMC+d35+SN50组、PBMC+d35+LPS组、PBMC+d35+LPS+SN50组和PBMC+d21+LPS组。取保存第1、21、35天(d1、d21、d35)的悬浮红细胞,离心,取上清,与新鲜分离的PBMC混合孵育20 h(不加库存红细胞各组用完全1640培养基培养),库存红细胞上清的体积为PBMC培养体系总体积的20%。加SN50各组于培养体系中加入核因子-κB特异性抑制剂SN50,培养4 h后,加LPS各组加入100 ng/L LPS刺激24 h,收集各组细胞培养上清,ELISA法检测肿瘤坏死因子-α(TNF-α)浓度的变化;收集PBMC与库存红细胞上清及LPS共同孵育各组的PBMC,免疫印迹法检测核因子-κB抑制剂-α(IκB-α)的蛋白水平及磷酸化水平的变化,以PBMC和PBMC+LPS组作为对照。结果:随库存红细胞保存时间的延长,PBMC中IκB-α的磷酸化水平逐渐升高,而蛋白水平随之逐渐下降;悬浮红细胞上清能增强LPS诱导下PBMC分泌TNF-α的作用,但能被SNS0抑制。结论:NF-κB信号通路参与了库存红细胞增强LPS诱导的PBMC炎症反应的作用,阻断NF-κB的活化可以抑制这一增强作用。Objective：To explore the effect and mechanism of banked red blood cells（RBCs） on enhanced secretion of inflammation cytokines of peripheral blood mononuclear cells （PBMCs） induced by lipopolysaccharide （LPS）. Methods：The supernatants were obtained from the stored RBCs for different time intervals （day 1, day 21 and day 35） respectively, and PBMCs from healthy donors were incubated in culture medium containing 20% supernatant of stored RBC for 24 hours. Then LPS was added, and cultivation was continued for 24 hours. After atotal of 48 hours incubation, the PBMCs were collected to determine LePta protein level and phosphorylation by Western blotting. PBMCs and PBMCs stimulated by LPS served as controls. SN50, a specific inhibitor for nuclear factor κB （NF-κB）, was added to the mixture of PBMCs and banked RBC supernatants, and incubated for 4 hours prior to LPS stimulation. Thereafter, the supernatants were collected and tumor necrosis factor-α （TNF-α） concentration was assayed by enzyme linked immunoadsorbent assay （ELISA） after 24-hour LPS stimulation. Results： The level of κB-α protein in incubated PBMCs was decreased and the level of IκB-α phosphorylation in incubated PBMCs was increased parallel with the duration of RBCs storage. TNFa secretion induced by LPS from incubated PBMCs was enhanced by supernatants of stored RBCs, and the effect was inhibited by SN50. Conclusions：Banked RBCs could enhance the inflammatory response of PBMCs induced by LPS, and NF-κB signal pathway is involved in the effect on the inflammatory response of PBMCs by banked red blood cell transfusion.

关 键 词：悬浮红细胞 单个核细胞 内毒素 核因子-ΚB 炎症反应 

分 类 号：R512.62[医药卫生—内科学]