LPS刺激后小鼠巨噬细胞Toll样受体(TLR)4及肿瘤坏死因子α表达的变化

摘要

Background Gram-negative septic shock is characterized by tissue and organ damage resulting from hyperproduction of cytokines and inflammatory mediators by the immune system in response to bacteria and LPS.One of the Toll-like receptors(TLRs.the family of mammalian proteins homologues of Drosophila Toll,play important roles in host defense),TLR4,mediates the responsiveness to LPS.It responds to consened structures within pathogens and actives macrophages,monocytes,and neutrophils to produce cytokines and inflammatory mediators.Here we investigated the expression of TLR4 and production of TNFα one of the most important inflammatory cytokines,in macrophages from peritoneal eavit of mice(Mφ)induced by LPS in vitro.Methods Mφ obtained from peritoneal cavity of BALB/C mice routinely were divided into six groups,where the cells were incubated with 0,0.001,0.01,0.1,1 and 10μg/ml of LPS(final concentrations)at 37℃ in 5%CO2/95% air respectively for 2h and cell-surface TLR4 was measured by flow cytometry.Simultaneously.TNFαin culture supernatant was detected by ELIsA．The experiments were repeated 3 times．Results ① The positive percentage of,TLR4 on the surface of Mφ stimulated with 0.01μg/ml of LPS was significantly higher than that of 0μgd group(P＜0.05＝.Except this,there was no difference between 0μg/ml group and other groups.However,the mean fluorescence intensity(MFIs) of TLR4 on the surface of Mφ were intensified with the increasing concentrations of LPS.Except that of 10μg/ml group.the MFIs of the treated groups were all significantly higher than that of 0μg／nrt group．② LPS induced a notable dose-dependent increase in TNFα secretion from Mφ and the production also decreased when LPS concentration was 10μg/m1.Conclusions Mφ respond to treatment of increasing concentrations of LPS by increased TLR4 expression and TNFα production.%革兰阴性菌败血症休克的发生主要是因为免疫系统对细菌LPS应答,产生过量细胞因子和炎性介质从而引起组织和器官损害.研究发现与果蝇Toll蛋白类似的哺乳动物Toll样受体家族在宿主防御中起重要作用,其中TLR4介导针对LPS的免疫应答.它们对病原体的保守结构产生应答并目激活单核细胞和中性粒细胞产生细胞因子和炎性介质,本研究观察LPS刺激后体外培养的小鼠腹腔巨噬细胞表面TLR4表达及最重要的炎性细胞因子之一的肿瘤坏死因子α分泌水平的变化.方法 常规方法从BALT/C小鼠腹腔中分离出巨噬细胞(Mψ),细胞分为6组,分别加入LPS使其终浓度为0、0.001、0.01、0.1、1、10μg/ml,5％CO2,37℃孵育2h.流式细胞术测定Mψ表面TLR4的表达.同时取培养液上清采用ELISA方法检测TNFα.实验重复三次.结果 ①经0.01μg/ml LPS刺激的Mψ TLR4-PE阳性率与0μg/ml LPS组相比,P<0.05.其它浓度刺激组TLR4阳性率则与对照组无差异,P>0.05.Mψ细胞TLR4 MFI随着LPS浓度的升高而增强,除了10μg/mlLPS组,其余各组MFI与对照组相比,P<0.05. ②随着LPS浓度增加,TNFα分泌量逐渐升高,呈剂量依赖关系.但当LPS浓度达10μg/ml时,TNFα分泌量下降.结论LPS能够诱导Mψ胞膜表面TLR4表达和TNF α分泌升高,并且在一定浓度范围内呈量-效关系.