关键词: 树突状细胞, 调节性, 骨髓单个核细胞, 细胞因子, 粒细胞-巨噬细胞集落刺激因子, 白细胞介素10, 转化生长因子β1, 免疫耐受, 体外培养, 鉴定

Abstract:

BACKGROUND: CD11clowCD45RBhigh regulatory dendritic cells (DCregs) have potential significance in participating the immune negative regulatory mechanism and clinical treatment. But it is still little about the induction and identification of human bone marrow-derived regulatory dendritic cells.
OBJECTIVE: To induce the human bone marrow-derived CD11clowCD45RBhigh regulatory dendritic cells and identify them from the aspects of cell morphology, immunological phenotype, the ability of phagocytosis and stimulating T lymphocytes proliferation in vitro.
METHODS: Mononuclear cells isolated from human bone marrow cells were induced and cultured with different cytokines in vitro. The cells were divided into three groups: (1)Regulatory dendritic cells group: regulatory dendritic cells were cultured for 7 days in RPMI 1640 culture medium containing granulocyte-macrophage colony-stimulating factor, interleukin-10 and transforming growth factor-β1, then stimulated with lipopolysaccharide for 24 hours. (2)Immature regulatory dendritic cells group: the immature regulatory dendritic cells were cultured for 8 days in RPMI 1640 culture medium containing granulocyte-macrophage colony-stimulating factor and interleukin-4. (3)Mature regulatory dendritic cells group: mature regulatory dendritic cells were cultured for 7 days in RPMI 1640 culture medium containing granulocyte-macrophage colony-stimulating factor and interleukin-4, and then stimulated with lipopolysaccharide for 24 hours to make them mature. The morphological phenotypes were observed under light and scanning microscope, the immunological phenotypes and ability of phagocytes were detected with flow cytometry, and the proliferative capacity of T cells stimulated by different subset of regulatory dendritic cells were examined by the Cell Count Kit-8.
RESULTS AND CONCLUSION: Mononuclear cells separated from human bone marrow cells differentiated into a new CD11clowCD45RBhigh subset of regulatory dendritic cells after cultured in the culture medium of RPMI 1640 containing granulocyte-macrophage colony-stimulating factor and interleukin-10 and transforming growth factor-β1 for 8 days, which has the typical morphology and immunological phenotype, and their immunological phenotype and immunological function had the characteristics of immature regulatory dendritic cells. The results showed that our method to culture the regulatory dendritic cells was feasible. The CD11clowCD45RBhigh regulatory dendritic cell is a new subset of dendritic cell, which has the powerful of phagocytosis function and weak ability to stimulate the proliferation of T lymphocytes. Here we establish an experimental basis for the researched on regulatory dendritic cells in immune tolerance.