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结合树突状细胞(DC)生物学特性,探讨抗P-选择素lectin-EGF功能域单抗(PsL-EGFmAb)对体外培养人DC成熟和功能干预调节的作用.通过SCF,GM-CSF,TGF-β1,Flt-3L及TNF-α体外培养体系,从脐血CD34+造血干细胞中诱导扩增获得DC,并于细胞成熟过程中用PsL-EGFmAb及辅以IL-10作为对照进行干预.分别观察和检测DC形态学及细胞活力,细胞表面分子HLA-DR,CD1a,CD11c,CD54,CD83,CD80,CD86,CD209(DC-SIGN)及CD62P,E,L(P-、E-、L-选择素)表达,细胞内活性氧(ROS)水平,及IL-12p35,p40 mRNA与NF-κBP50,P65 mRNA表达,培养上清液中IL-12p70分泌含量,以及DC体外对T淋巴细胞刺激能力,以此分析PsL-EGFmAb对DC成熟与功能的干预状况.结果显示,未成熟DC高表达属模式识别受体的C型凝集素DC-SIGN外,且胞内蓄积适量ROS,具备了细胞吞噬能力.成熟DC除仍高表达DC-SIGN,伴随细胞内NF-κB基因明显表达,其表面黏附共刺激分子CD11c,CD83,CD80,CD86表达上调,且细胞因子IL-12合成分泌增加,并具明显的体外刺激T淋巴细胞增殖能力,符合于抗原提呈细胞特征.此外,未成熟和成熟DC基本不表达P-,E-选择素,而分别高表达和低表达L-选择素.进一步发现,PsL-EGFmAb较对照IL-10对DC表面DC-SIGN表达有抑制作用;也能抑制细胞内NF-κB基因表达,并相应抑制或下调DC黏附共刺激分子CD11c,CD83,CD80,CD86及HLA-DR表达,抑制IL-12基因转录及其合成分泌,以及抑制DC体外刺激T细胞增殖的能力.上述结果表明,PsL-EGFmAb对DC分化成熟及功能具有抑制作用,提示此作用与其抑制作为DC模式识别受体及功能分子DC-SIGN有关,并可能是通过影响NF-κB信号途径起作用.
To investigate the effect of anti-P-selectin lectin-EGF mAb on the regulation of human DC maturation and functional intervention in vitro by using the biological characteristics of dendritic cells (DCs) TGF-β1, Flt-3L and TNF-α in vitro, DCs were induced from cord blood CD34 + hematopoietic stem cells and were intervened by PsL-EGFmAb and IL-10 supplemented in the process of cell maturation respectively The morphology and cell viability of DCs were observed and detected. The cell surface molecules such as HLA-DR, CD1a, CD11c, CD54, CD83, CD80, CD86, CD209 and CD62P, E, Selectin expression, intracellular reactive oxygen species (ROS) level, IL-12p35, p40 mRNA and NF-κBP50, P65 mRNA expression in culture supernatant IL-12p70 secretion, and DC in vitro stimulation of T lymphocytes , In order to analyze the intervention status of PsL-EGFmAb on the maturation and function of DC.The results showed that immature DCs were highly expressed outside the C-type lectin DC-SIGN which is a pattern recognition receptor, and the intracellular accumulation of appropriate ROS, with phagocytosis Ability DCs still express DC-SIGN with high expression of CD11c, CD83, CD80 and CD86 on the surface of DCs, with the expression of NF-κB gene in cells significantly increased IL-12 secretion increased, and significantly stimulated the proliferation of T lymphocytes in vitro, in line with the characteristics of antigen-presenting cells.In addition, immature and mature DC basically do not express P-, E-selectin, respectively, and were highly expressed And low expression of L-selectin.It was further found that PsL-EGFmAb could inhibit the expression of DC-SIGN on the surface of DC as compared with the control IL-10, and also inhibit the expression of NF-κB in the cells and correspondingly inhibit or downregulate DC adhesion co-stimulation The expression of CD11c, CD83, CD80, CD86 and HLA-DR could inhibit the transcription of IL-12 gene and its synthesis and secretion as well as the ability of DC to stimulate the proliferation of T cells in vitro.The above results indicated that PsL- Inhibition, suggesting that this effect and its inhibition as a DC pattern recognition receptor and functional molecules DC-SIGN-related, and may be through the impact of NF-κB signaling pathway.