CD11c^(low)CD45RB^(high)调节性树突状细胞的诱导培养及鉴定  

Induction and identification of CD11c^(low)CD45RB^(high) regulatory dendritic cells

作　　者：刘苍春[1] 黄一虹[1] 冯洒然[2] 谷红红[1] 曾令宇[1] 李德鹏 徐开林[1] 

机构地区：[1]徐州医学院附属医院血液科,江苏省徐州市221002 [2]徐州市中心医院,江苏省徐州市221002

出　　处：《中国组织工程研究》2012年第53期9987-9993,共7页Chinese Journal of Tissue Engineering Research

基　　金：徐州市科技计划项目(XF10C065)资助~~

摘　　要：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7d后,用脂多糖刺激24h。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8d。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7d后,予脂多糖刺激24h,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8d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。BACKGROUND： CD11c low CD45RB high 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 CD11c low CD45RB high 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 ofT 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 CD11c low CD45RB high subset of regulatory dendritic cells after cultured in the

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 

分 类 号：R318[医药卫生—生物医学工程]