山东医药2016，Vol.56Issue(10)：13-16,4.DOI:10.3969/j.issn.1002-266X.2016.10.005
小鼠骨髓来源调节性树突状细胞的体外诱导分化及鉴定
Induced differentiation and identification of mouse bone marrow-derived regulatory dendritic cells in vitro
摘要
Abstract
Objective To establish a method for the induction and amplification of regulatory dendritic cells ( DCreg) derived from mouse bone marrow in vitro, and to identify their biological and functional characteristics.Methods The bone marrow mononuclear cells ( BM-MNCs) were induced to differentiate into DCreg by recombinant mouse IL-4 and re-combinant murine granulocyte-macrophage colony-stimulating factor ( rmGM-CSF ) .The cells collected on the sixth day contained a large number of DCreg.A portion of cells were cultured with lipopolysaccharide ( LPS) for another 2 days to a-chieve the mature DC (mDC).The morphology of DCreg was observed by inverted microscope.The expression of CD40, CD11b and CD86 were detected by flow cytometry.The splenic CD8 +T cells were randomly divided into three groups:groups A, B and C.The cells in the group A and group B were incubated with DCreg or mDC at different ratios (5:1, 10:1, 20:1) of mixture to culture for 4 days, and cells in the group C were not treated.We detected the proliferation of CD8 +T cells by the CFSE labeling.The expression of perforin ( Prf1) and GzmB mRNA in CD8 +T cells was detected by real-time PCR.Results Freshly isolated MSCs were round with small volume.When induced for 6 days, much more cell colonies were found, and cells became larger with the increased cell surface projections.The positive cell percentage of CD11b was (96.1 ±2.7)%.Compared with mDC, the positive expression rates of CD40 and CD86 in DCreg were lower (all P<0.05).Compared with group B, the proliferation rate of group A was significantly decreased (all P<0.05). Compared with group C and group B, the expression of Prf1, GzmB mRNA of group A was reduced (all P<0.05).Com-pared with group C, the expression of GzmB mRNA in the group B was increased (all P<0.05).Conclusions rmGM-CSF combined with rmIL-4 can induce the differentiation of BM-MNCs into DCreg successfully, which can obtain a large number of highly purified DCreg with immune suppressive function.关键词
调节性树突状细胞/器官移植/免疫耐受/细胞培养/穿孔素/颗粒酶BKey words
regulatory dendritic cells/organ transplantation/immune tolerance/cell culture/perforin/granzyme B分类
医药卫生引用本文复制引用
王进京,袁晶华,乔磊,刘义,张晓宁,李克秋,李光..小鼠骨髓来源调节性树突状细胞的体外诱导分化及鉴定[J].山东医药,2016,56(10):13-16,4.基金项目
国家高技术研究发展计划项目(2012AA021003). （2012AA021003）
