黑素瘤细胞培养上清液对人单核细胞体外IL-12产生和CD14、CD1a表达的影响

The effects of melanoma culture supernatants on production of IL-12 and expression of CD14 and CD1a by human monocytes in vitro

目的探讨M14黑素瘤细胞培养上清液(MCS)在体外对人外周血单核细胞分泌白介素12 (IL-12)以及单核细胞向树突细胞分化的影响。方法分离并获得人外周血单核细胞,添加不同量的MCS与单核细胞共培养,用酶联免疫吸附法(ELISA)检测单核细胞IL-12的分泌情况;将MCS与单核细胞连续5d共培养,用流式细胞仪分析单核细胞表面CD14、CD1a的表达情况。结果MCS对单核细胞分泌活性的影响呈浓度依赖关系,随MCS添加量的逐渐增加(25-100μL),单核细胞分泌IL-12的量则呈逐渐降低趋势(158.40±21.37 pg/mL-56.90±15.60 pg/mL,P<0.05);同时MCS妨碍单核细胞由CD14表型(5.47%±1.00%-17.87%±2.40%,P<0.01)向树突细胞CD1a表型(41.00%±2.12%-20.00%±4.24%,P<0.01)转化;且MCS抑制单核细胞IL-12分泌和抑制单核细胞向树突细胞表型转化的作用均能被细胞外信号调节激酶(ERK)特异性抑制剂(PD98059)所逆转(分别为:172.63±8.88 pg/mL,P<0.05; 8.67%±0.47%,P<0.05;36.67%±0.71%,P<0.05)。结论MCS可能通过分泌ERK刺激因子对单核细胞分泌IL-12p40和单核细胞分化起到抑制作用。

Objective To investigate the effects of melanoma culture supernatants （ MCS ） on the monocytic interleukin-12 （ IL-12 ） production and on the monocyte-derived dendritic cell （ DC ） differentiation in vitro. Methods Human peripheral blood monocytes were isolated and the purity of the monocytes was 〉 85%. The concentrated MCS in different amount was added to the IFN-γ （ 100 pg/mL ） and LPS （ 10 ng/mL ） enriched culture media. The IL-12 production by monocytes was determined by the enzymelinked immunosorbent assay （ ELISA ）. The expression of CD14 and CDla was analyzed by flow cytometry 5 days after the monocytes were co-cultured with MCS. Results The production of monocytic IL-12 was down-regulated by MCS in a dose dependent manner. The amount of IL-12 from monocytes decreased along with an increased dose （ 25 - 100 μL ） of MCS applied in the reaction. It was also observed that the differentiation from CD14 expressing monocytes to CD1a dendritic cells was impaired by MCS. The ability of MCS to inhibit the production of IL-12 by monocytes and to suppress the differentiation of monocytes to dendritic cells in vitro could be disrupted by PD98059,. an ERK specific inhibitor. Conclusions MCS appears to inhibit IL-12p40 production by monocytes and inhibit differentiation of monocytes in vitro via secretion of ERK stimulating factor, The inhibitory factors in MCS and their chemical natures need further research.