LPS耐受巨噬细胞致炎和抗炎细胞因子表达和分泌的特点

目的：分析巨噬细胞LPS耐受形成过程中致炎和抗炎细胞因子（TNF-α、IL-6和IL-10）释放的特点和规律,探讨其在巨噬细胞建立LPS耐受机制中的作用.方法：培养小鼠腹腔巨噬细胞系RAW264.7 细胞.实验分为两部分：①LPS刺激细胞不同时间（4 h,8 h,20 h）或用PBS培养细胞4 h;②LPS耐受实验（LPS预刺激巨噬细胞20 h后,换液并洗去LPS,再次加入LPS刺激4 h）.ELISA法检测细胞培养上清中TNF-α、IL-6和IL-10的浓度,RT-PCR法相对定量分析巨噬细胞TNF-α和IL-10 mRNA 表达水平.结果：细胞培养上清中TNF-α、IL-6和IL-10的浓度随着LPS刺激时间的延长而增加;LPS刺激20 h后建立耐受的巨噬细胞再次受到LPS打击时,其TNF-α释放量低于非耐受细胞,而IL-6和IL-10释放量则大幅增加,与TNF-α释放减少截然相反.TNF-α和IL-10mRNA 表达水平与其蛋白分泌量呈现相似的变化规律.结论：巨噬细胞对LPS的耐受建立在致炎因子和抗炎因子共同作用的基础上;LPS预刺激细胞再次受到LPS打击时,抑炎因子表达和分泌大幅增加是介导巨噬细胞对LPS耐受的重要机制.

Expression and secretion pattern of proinflammatory and anti-inflammatory cytokines in lipopolysaccharide-tolerized macrophages

Objective： To analyze the patterns of TNF, IL-6 and IL-10 secretion and their mRNA expression in endotoxin （LPS）-stimulated and LPS tolerant macrophages, and to explore the role of proinflammatory and anti-inflammatory cytokines in hyporesponsive macrophages to LPS re-stimulation. Methods： In the first set of experiments,macrophages-like ceils （RAW264.7） were incubated with LPS for 0,4,8 and 20 hours. In the second set of experiments,RAW264.7 cells were pretreated with LPS for 20 hours,then the culture medium was discarded and the cells were restimulated with LPS for 4 hours. Contents of TNF-α,IL-6 and IL-10 secretions were measured in the supernatant using enzyme-linked immunosorbent assay. The expression of TNF-α and IL-10 mRNA was determined by means of semiquantitative RT-PCR. Results： Release of TNF-α,IL-6 and II.-10 was gradually increased along with LPS stimulation time. TNF-α release was reduced from LPS tolerant maerophages （pretreated with LPS for 20 hours）, whereas IL-6 and IL-10 release was augmented in response to LPS restimulation. Change in TNF-α and IL-10 mRNA expression exhibited the same pattern as that of the secretion of their proteins. Conclusions： Macrophages tolerance to LPS is induced by coordinated function of proinflammatory and anti-inflammatory cytokines. When LPS-tolerized macrophages is restimulated with LPS,augmented IL-10 expression and release are the underlying mechanism of tolerance of macrophages to LPS.