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本研究探讨甘露聚糖结合凝集素(MBL)对细菌脂多糖(LPS)诱导的人外周血单核细胞来源树突状细胞(DC)成熟的机制。从健康成人外周血分离单核细胞(Mo),用常规方法体外诱导未成熟DC(imDC),FACS分析MBL与imDC的结合,并进一步分析MBL对LPS结合imDC的影响;ELISA和Western blot分析MBL与可溶性的TLR4胞外段蛋白(sTLR4)结合;Western blot分析NF-κB的细胞核移位。结果表明,在钙离子存在下,MBL能够直接与imDC结合,sTLR4和LPS可竞争性抑制MBL与imDC的结合。Western blot与ELISA分析结果显示,MBL能够以浓度依赖的方式结合sTLR4蛋白。FACS分析结果进一步表明,MBL通过结合imDC竞争性抑制LPS与imDC的结合。Western blot分析结果亦显示,MBL对LPS诱导的NF-κB细胞核移位有显著的抑制作用。结论:MBL可通过结合表达在imDC的TLR4分子竞争性抑制LPS结合imDC,从而抑制LPS诱导imDC成熟,提示MBL能够通过配体结合调控DC分化成熟。本研究为阐明MBL抑制LPS诱导DC成熟的机制提供了较好的实验依据。
This study was aimed to investigate the mechanism of mannan-binding lectin (MBL) maturation induced by bacterial lipopolysaccharide (LPS) on dendritic cells (DCs) derived from human peripheral blood mononuclear cells. Monocytes (Mo) were isolated from peripheral blood of healthy adults, immature DCs (imDC) were induced in vitro by conventional methods, the binding of MBL to imDC was analyzed by FACS, and the effect of MBL on LPS-bound imDC was further analyzed; MBL And soluble TLR4 extracellular domain protein (sTLR4) binding; Western blot analysis of NF-κB nuclear translocation. The results showed that MBL could directly bind to imDC in the presence of calcium ions, and sTLR4 and LPS could competitively inhibit the binding of MBL to imDC. Western blot and ELISA analysis showed that MBL could bind sTLR4 protein in a concentration-dependent manner. FACS analysis further demonstrated that MBL competitively inhibited the binding of LPS to imDC by binding to imDC. Western blot analysis also showed that MBL significantly inhibited LPS-induced nuclear translocation of NF-κB. CONCLUSION: MBL can competitively inhibit LPS-bound imDC by binding to TLR4 expressed in imDC, thereby inhibiting LPS-induced imDC maturation, suggesting that MBL can regulate DC maturation via ligand binding. This study provides a good experimental evidence for elucidating the mechanism by which MBL inhibits LPS-induced DC maturation.