目的研究负载滋养层细胞抗原对小鼠髓源性树突状细胞(DC)分化成熟过程的影响,获得致耐受性DC。方法体外使用粒细胞巨细胞集落刺激因子(GM-CSV)诱导小鼠骨髓细胞定向分化、经LPS刺激获得成熟DC;通过外胎盘锥组织块培养法获得滋养层细胞,制备可溶性抗原,加入DC培养体系。流式细胞术检测DC表面共刺激分子及MHC-Ⅱ的表达,ELISA法检测DC分泌IL-10和IL-12的浓度,混合淋巴细胞培养评估DC刺激同种T细胞增殖、活化的功能。结果成熟DC表型为CD4d域CD80恸CD86^high油MHC-Ⅱ“曲,分泌大量的IL-12和极少量的IL-10,体外能有效刺激T细胞的增殖;负载滋养层细胞抗原的Dc表型为CD40^mid。CDS0hCD86^low MHC-Ⅱ^low,在分泌大量IL-12的同时IL-10也明显升高,不能有效刺激T细胞增殖,并使T细胞分泌细胞因子呈现明显Th2偏倚。结论负载滋养层细胞抗原后的DC表面共刺激分子及MHC一Ⅱ表达降低,刺激T细胞增殖能力下降;其自分泌和促使T细胞旁分泌的细胞因子呈现Th2偏倚,是一种耐受性DC。
Objective To study the phenotype and function of murine myeloid dendritic cells (DCs) loaded with trophoblastic cell antigens. Methods Murine bone sorrow cells were cultured with granulocyte macrophage colony stimulating factor(GM-CSF) in vitro and induced to mature DCs by LPS. Murine trophoblastic cells were obtained by culture of ectoplacenta cone (EPC) tissue and made into soluble antigens. After loading with trophoblastic cell antigen, the expression of costimulatory molecules and MHC-Ⅱ on DCs were detected by FACS; the concentration of IL-10 and IL-12 secreted by DCs were detected by ELISA; the function of DCs was evaluated by mixed lymphocyte reaction. Results the phenotype of DCs loaded with trophoblastic cell antigen was CD40^mid CD80^low CD86^low MI-IC-Ⅱ^low. The secretion of IL- 10 was higher than that of mature DCs while the secretion of IL-12 was similar to that of mature DCs, which led to a higher IL-10/IL-12 ratio. In mixed lym- phocyte reaction, the DCs could not induce allogeneic T cells proliferation effectively, which was associated with the inhibition of Thl type cytokines (IFN-γ) production and promotion of Th2 type cytokines (IL-10 and IL-4). Conclusion Murine myeloid DCs loaded with trophoblastic cell antigen have different phenotype characterized by lower costimulation molecular and MHC-II expression. These DCS are tolemgenic DCs which could induce allogeneic T cells hyporesponsiveness and the autoerine and paracrine Th2 cytokine bias.