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

作　　者：王进京[1] 袁晶华 乔磊[1] 刘义[1] 张晓宁[1] 李克秋[1] 李光[1] 

机构地区：[1]天津医科大学基础医学院,天津300070

出　　处：《山东医药》2016年第10期13-16,共4页Shandong Medical Journal

基　　金：国家高技术研究发展计划项目(2012AA021003)

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(rm IL-4)及重组小鼠粒-巨噬细胞集落刺激因子(rm GM-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(m DC)。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、m DC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(Gzm B)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m DC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、Gzm B mRNA表达量降低(P均<0.05),与C组比较,B组Gzm B mRNA表达量升高(P均<0.05)。结论 rm GM-CSF联合rm IL-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 recombinant murine granulocyte-macrophage colony-stimulating factor（ rm GM-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 achieve the mature DC（ m DC）. The morphology of DCreg was observed by inverted microscope. The expression of CD40,CD11 b 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 m DC 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 Gzm B 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 CD11 b was（ 96. 1 ± 2. 7） %. Compared with m DC,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,Gzm B mRNA of group A was reduced（ all P 〈0. 05）. Compared with group C,the expression of Gzm B mRNA in the group B was increased（ all P 〈0. 05）. Conclusions rm GMCSF combined with rm IL-4 can induce the differentiation of BM-MNCs into DCreg successfully,which can obtain a large number of highly purified

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B 

分 类 号：R392.4[医药卫生—免疫学]