摘要
目的:探讨内毒素耐受和拟胆碱药物卡巴胆碱对巨噬细胞肿瘤坏死因子-α(TNF-α)分泌的调节作用。方法:①分离小鼠腹腔巨噬细胞,将其与内毒素(LPS)作用不同时间(0、2、4、8 h);②小鼠腹腔巨噬细胞分为3组:空白对照组(RPMI1640常规培养),LPS耐受组(LPS预刺激20 h,再用LPS刺激4 h)和LPS不耐受组(RPMI1640常规培养20 h后,再加LPS刺激4 h);③分离大鼠腹腔巨噬细胞,分为3组:空白对照组、LPS刺激组及卡巴胆碱+LPS刺激组(卡巴胆碱20μmol/L预处理后LPS刺激)。各组LPS的剂量均为100 ng/ml。收集各组细胞培养上清液及细胞裂解液,用酶联免疫分析(ELISA)方法检测TNF-α浓度。结果:LPS预刺激巨噬细胞20 h后再次用LPS刺激时,其TNF-α分泌量不增加,与空白对照组接近,同时细胞内TNF-α含量也不增加。未经LPS预刺激的巨噬细胞在受到LPS刺激后TNF-α分泌量大幅增加,同时细胞内TNF-α含量也伴随增加,即细胞分泌和贮存的TNF-α量的变化一致。卡巴胆碱预处理巨噬细胞受内毒素刺激后TNF-α分泌量低于单纯LPS刺激组细胞,但细胞内的TNF-α含量明显高于空白对照组(P<0.01),与单纯LPS刺激组接近。结论:巨噬细胞建立LPS耐受机制或受到拟胆碱药物作用后,再次受到LPS刺激后均表现为TNF-α分泌的减少,但细胞内TNF-α量变化规律不一致。提示内毒素耐受和胆碱能抗炎所致巨噬细胞炎症反应特性改变的机制不同。
Objective：To investigate the difference in regulating tumor necrosis factor -α （TNF-α） secretion between endotoxin tolerance and cholinergic drug-carbachol pre-treated maerophages. Methods： （1） The peritoneal macrophages were harvested from male mice, and they were exposed to endotoxin （LPS） for 0, 2, 4, and 8 hours. （2）The isolated peritoneal macrophages of mice were divided into 3 groups： control group （RPMI1640 routine culture）, LPS pre-treatment group （pre-exposure to LPS followed by LPS stimulation）, and LPS stimulation group. （3） The peritoneal macrophages of rats were isolated and were divided into 3 groups： control, LPS stimula- tion, and carbachol pre-treatment （LPS stimulation following pre-exposure to carbachol） groups. Lysate of macro- phages and cell supernatants in all experiments were harvested to determine the content of TNF-α by enzyme- linked immunoadsorbent assay （ELISA） method. Results： The amount of TNF-α secretion in LPS pre-treated macrophages was not increased following being stimulated by LPS again, and no difference was observed between LPS pre-treated and control groups. No increase in TNF-α content was observed in LPS pre-treated macrophages. But for the LPS untreated macrophages, the amount of secreted TNF-α was increased dramatically accompanying with an increase in TNF-α content in the macrophages after being stimulated by LPS, showing that the quantity of both secreted and reserved TNF-α was significantly high. For carbachol pre-treated cells, the amount of TNF-α secretion was lower than in that of carbachol untreated cells, but the content of TNF-α in cells was higher than that in the control （P〈0.01）, and it was close to that in the LPS stimulating cells. Conclusions After being stimula- ted by LPS, the secretion of TNF-α from both endotoxin tolerant and cholinergic pre-treated macrophages is reduced, but there is a discordance in TNF-α content in the two groups. It is suggested that in macrophages, the mechanisms of
出处
《感染．炎症．修复》
2011年第3期144-148,共5页
Infection Inflammation Repair
基金
国家自然科学基金面上项目(30770844)
