内毒素耐受和胆碱能受体兴奋对巨噬细胞贮存和分泌TNFα的影响.docVIP

内毒素耐受和胆碱能受体兴奋对巨噬细胞贮存和分泌TNFα的影响 解放军总医院第一附属医院全军烧伤研究所 吕 艺 周国勇 胡 森 【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TNFα分泌的调节作用。方法 ①分离小鼠腹腔巨噬细胞，将其与LPS作用不同时间； ②小鼠腹腔巨噬细胞分为三组：空白对照组（RPMI1640常规培养），LPS耐受+刺激组（LPS预刺激20h,再用LPS刺激4h）和LPS刺激组（RPMI1640常规培养20h 后，再加LPS刺激4h）；③分离大鼠腹腔巨噬细胞，分为三组：空白对照组、LPS刺激组及卡巴胆碱预处理后LPS刺激组。收集各组细胞培养上清液及细胞裂解液，用ELISA方法检测TNFα浓度。结果 LPS预刺激巨噬细胞20h后再次用LPS刺激时，其TNFα分泌量不增加，与对照组接近，同时细胞内TNFα含量也不增加。未经LPS预刺激的巨噬细胞在受到LPS刺激后TNFα分泌量大幅增加，同时细胞内TNFα含量也伴随增加，即细胞分泌和贮存的TNFα量变化一致。卡巴胆碱预处理巨噬细胞受内毒素刺激后TNFα分泌量低于单纯内毒素刺激组细胞，但细胞内的TNFα含量高于对照组，与单纯内毒素刺激组接近。 结论 巨噬细胞建立内毒素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TNFα分泌的减少，但细胞内TNFα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。 【关键词】巨噬细胞；内毒素耐受； 拟胆碱药；肿瘤坏死因子 【Abstract】Objective To investigate the difference of regulating TNFαsecretion between endotoxin tolerance and cholinergic drug--carbachol pre-treated macrophages. Methods ① the peritoneal macrophages were obtained from male mice, which were exposure to endotoxin(LPS) for 0, 2, 4, and 8 hours; ② the isolated peritoneal macrophages were divided into 3 groups: control(RPMI1640 routine culture), LPS pre-treatment(pre-exposure to LPS followed by LPS stimulation, and LPS stimulation; ③ the peritoneal macrophages of rats were isolated and were divided into 3 groups: Control, LPS stimulation, and carbachol pre-treatment (LPS stimulation following pre-exposure to carbachol) groups. Lysate of macrophages in experiment and cell supernatants in all experiments were harvested to determined the content of TNFα by ELISA method. Results The amount of TNFα secretion in LPS pre-treated macrophages was not increased following stimulated with LPS again, and no difference was observed between LPS pre-treated and control groups. No increase in TNFα content was observed in LPS pre-treated macrophages. But for the LPS untreated macrophages, the amount of secreted TNFα was increased dramatically and accompanied with the increase of TNFαcontent in the macrophages after being stimulated by LPS, namely both secreted and reserved TNFα were signif