羧甲基茯苓多糖对人外周血源性树突状细胞SOCS-1基因甲基化及体外成熟的影响

Carboxymethytl pachymaram induces the methylation of SOCS-1 gene and the maturation of human monocyte-derived dendritic cells

目的：通过检测羧甲基茯苓多糖（ carboxymethytl pachymaram ，CMP）处理后人外周血源性树突状细胞（dendritic cells，DC）中细胞因子信号转导抑制分子-1（SOCS-1）基因甲基化水平及表达情况，探讨 CMP 对 DC 体外成熟的影响。方法通过重组人粒细胞-巨噬细胞集落刺激因子（rhGM-CSF）和IL-4体外诱导人外周血源性单核细胞源DC，并促进成熟，分别加入不同浓度的CMP （终浓度为10、50、100 mg/L），应用甲基化特异性PCR（ methylation specific PCR ，MSP）和real-time PCR法分别分析SOCS-1基因甲基化水平及表达情况；应用流式细胞术、混合淋巴细胞反应（ MLR）和ELISA分别检测DC的表面标志、刺激淋巴细胞增殖能力和IL-12分泌的变化。结果经CMP刺激后DC中SOCS-1基因甲基化水平明显增高，SOCS-1基因表达水平显著降低，表面标志（ CD80、CD83、CD86、HLA-DR）、刺激淋巴细胞增殖能力及IL-12的分泌水平均上调，且呈剂量依赖趋势。50 mg/L和100 mg/L CMP刺激组SOCS-1基因甲基化水平、IL-12分泌量及刺激淋巴细胞增殖指数均显著高于对照组，SOCS-1基因表达水平显著低于对照组。100 mg/L CMP刺激组CD80、CD83和HLA-DR表达水平显著高于对照组。结论 CMP能促进人外周血源性DC中SOCS-1基因甲基化，减少SOCS-1基因表达，进而促进其体外成熟。

Objective To investigate the effects of carboxymethytl pachymaram （ CMP ） on the methylation of SOCS-1 （suppressor of cytokine signaling-1） gene and the in vitro maturation of human mono-cyte-derived dendritic cells （DCs）.Methods Human DCs were induced from the peripheral blood mono-cytes in vitro with the treatment of recombined human GM-CSF and interleukin-4 （ IL-4 ） and cultured with different concentrations of CMP （10, 50, and 100 mg/L）.The methylation and expression of SOCS-1 gene were analyzed by methylation-specific polymerase chain reaction （MSP） and real-time PCR, respectively. The phenotypic markers of DCs were detected by flow cytometry .Mixed lymphocyte reaction （ MLR） and ELISA were performed to measure the lymphocyte proliferation induced by DCs and IL-12 secretion by DCs . Results CMP promoted the methylation of SOCS-1 gene, but inhibited the expression of SOCS-1 gene in dendritic cells at the concentrations of 50 mg/L and 100 mg/L.The expression of phenotypic markers （CD80, CD83, CD86 and HLA-DR）, IL-12 secretion and lymphocyte proliferation induced by DCs were significantly enhanced in a dose dependent manner with the treatment of CMP .Compared with control group , the levels of methylated SOCS-1 gene and IL-12 and the lymphocyte proliferation index were increased upon the stimulation with 50 mg/L and 100 mg/L of CMP （P〈0.01）, but the expression of SOCS-1 gene was de-creased.The expression of CD80, CD83 and HLA-DR on DCs in the presence of 100 mg/L of CMP were higher than those of control group （P〈0.05）.Conclusion CMP could induce the methylation of SOCS-1 gene and the maturation of DCs derived from peripheral blood monocytes .