小鼠骨髓来源调节性树突状细胞的体外诱导分化及鉴定

摘要

目的 建立小鼠骨髓来源调节性树突状细胞(DCreg)体外诱导培养和扩增的方法,并进行生物学及功能鉴定.方法 取小鼠骨髓单个核细胞(BM-MNCs),用重组小鼠白细胞介素4(rmIL-4)及重组小鼠粒-巨噬细胞集落刺激因子(rmGM-CSF)诱导分化,第6天细胞中含大量DCreg;取部分细胞加脂多糖(LPS),继续培养2 d(第8天)分化为成熟DC(mDC).观察培养过程中细胞形态变化,流式细胞术检测DCreg特异性表面分子CD11b和共刺激分子CD40、CD86.取小鼠脾脏分离的CD8+T细胞,随机分为3组;A、B组将细胞分别与DCreg、mDC以5:1、10:1、20:1混合培养4 d,C组细胞不处理.采用CFSE标记法检测CD8+T细胞增殖情况,Real-time PCR检测CD8+T细胞免疫功能分子穿孔素(Prf1)和颗粒酶B(GzmB)mRNA.结果 新分离的BM-MNCs呈圆形,体积较小;诱导第6天细胞集落数量增多,体积增大,细胞表面突起增多,CD11b阳性细胞比例达96.1%±2.7%.与mDC相比,DCreg中CD40、CD86表达阳性率降低(P均<0.05).与B组比较,A组细胞增殖率降低(P均<0.05);与B、C组比较,A组Prf1、GzmB mRNA表达量降低(P均<0.05),与C组比较,B组GzmB mRNA表达量升高(P均<0.05).结论 rmGM-CSF联合rmIL-4可成功诱导小鼠BM-MNCs向DCreg分化,且纯度高,并具有免疫抑制功能.%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.