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目的 研究脂多糖 (LPS)的跨膜信号传导分子TLR4和MD 2在巨噬细胞内毒素耐受发生中的调节变化和作用。方法 分离小鼠腹腔巨噬细胞 ,活化组直接以 1mg/LLPS刺激一定时间 ,耐受组先用不同剂量LPS预处理 2 0h ,再用 1mg/LLPS刺激。利用逆转录 聚合酶链反应(RT PCR)测定各基因的转录调节变化。结果 在活化组细胞LPS刺激可显著诱导肿瘤坏死因子(TNF) α的基因转录和蛋白分泌 ,但是LPS预处理可使耐受组细胞TNF α的mRNA和蛋白水平均明显低于活化组 (P <0 .0 1) ;耐受组细胞中TLR4的基因表达水平也比活化组明显低 (P <0 .0 1) ,但MD 2的基因表达水平不受LPS刺激的影响。结论 内毒素耐受的巨噬细胞表现为抑制的炎症细胞因子产生 ,TLR4基因的表达下调可能是内毒素耐受发生的机制之一。
Objective To investigate the regulatory effect of lipopolysaccharide (LPS) transmembrane signal transduction molecules TLR4 and MD2 on macrophage endotoxin tolerance. Methods Mouse peritoneal macrophages were isolated. The activated group was stimulated with 1mg / LLPS for a certain time. The tolerated group was pretreated with different doses of LPS for 20h and then stimulated with 1mg / L LLPS. Transcriptional regulation of each gene was determined by reverse transcription polymerase chain reaction (RT PCR). Results LPS stimulated TNF-α gene transcription and protein secretion in LPS-treated cells, but LPS pretreatment significantly decreased TNFα mRNA and protein levels in LPS-treated cells (P < 0. 01). The gene expression of TLR4 in tolerant group was also significantly lower than that in activated group (P <0.01), but the gene expression of MD 2 was not affected by LPS stimulation. Conclusion Endotoxin-resistant macrophages exhibit suppressed cytokine production. Down-regulation of TLR4 gene may be one of the mechanisms of endotoxin tolerance.