CD11clowCD45RBhigh调节性树突状细胞的诱导培养及鉴定

背景：新近发现CD11clowCD45RBhigh调节性树突状细胞在参与免疫负向调控机制和临床治疗上有潜在的意义。目前对于健康人骨髓来源的调节性树突状细胞的体外诱导培养及鉴定的研究较少。目的：体外培养正常人骨髓来源的具有CD11clowCD45RBhigh表型的调节性树突状细胞,从细胞形态、免疫表型、吞噬功能及刺激T淋巴细胞增殖能力等方面对其进行鉴定。方法：分离正常人骨髓单个核细胞分别在不同的培养体系中进行体外诱导培养,实验分3组：①调节性树突状细胞组,在含粒细胞-巨噬细胞集落刺激因子,白细胞介素10,转化生长因子β1的1640培养基中培养7 d后,用脂多糖刺激24 h。②未成熟树突状细胞组,用含粒细胞-巨噬细胞集落刺激因子,白细胞介素4的1640培养基培养8 d。③成熟树突状细胞组,用含粒细胞-巨噬细胞集落刺激因子,白细胞介素4的1640培养基培养7 d后,予脂多糖刺激24 h,使其成熟。光镜和扫描电镜观察树突状细胞的形态,流式细胞仪检测其免疫表型以及吞噬能力,CCK-8法检测其刺激异基因淋巴细胞增殖能力。结果与结论：①人骨髓源单个核细胞在联合应用粒细胞-巨噬细胞集落刺激因子,白细胞介素10,转化生长因子β1培养8 d后获得的细胞具有调节性树突状细胞的典型特征和免疫表型,说明该实验建立的培养调节性树突状细胞的方法是切实可行的。②CD11clowCD45RBhigh调节性树突状细胞这种新亚群具有较强的吞噬能力,刺激T淋巴细胞增殖的能力较弱,为调节性树突状细胞诱导免疫耐受的进一步研究奠定实验基础。

Induction and identification of CD11clowCD45RBhigh regulatory dendritic cells

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.