小鼠骨髓来源调节性树突状细胞的体外培养及鉴定  

Induction and Identification of Mouse Bone Marrow-Derived Regulatory Dendritic Cells

作　　者：谷红红[1] 黄一虹[1,2] 武家庆[2] 刘苍春[1] 曾令宇[1] 李德鹏 李振宇[1] 徐开林[1] 

机构地区：[1]徐州医学院附属医院血液科,江苏徐州221002 [2]徐州医学院附属第三医院血液科,221003

出　　处：《国际输血及血液学杂志》2014年第1期5-10,共6页International Journal of Blood Transfusion and Hematology

基　　金：江苏省高校自然科学基金（07KJD320224）;徐州市科技计划项目（XMl38040,XZZDll49）

摘　　要：目的体外诱导培养小鼠骨髓来源调节性树突状细胞（DCreg），并从其细胞形态、免疫表型、功能特征等对其进行鉴定。方法无菌条件下取C57BL／6小鼠的骨髓单个核细胞，按体外诱导培养的培养体系不同，将其分为3组：未成熟DC（imDC）组（N=16），用含重组鼠粒-巨噬细胞集落刺激因子（rmGM-CSF）20ng／mL，重组鼠白细胞介素-4（rmlL-4）10ng／mL的RPMI1640培养基诱导培养7d；②成熟DC（mDC）组，用含rmGM-CSF20ng／mL，rmIL-410ng／mL的RPMI1640培养基培养6d，予细菌脂多糖（LPS）刺激24h，使其成熟；③DCreg组：用含rmGM—CSF20ng／mL，II。一1020ng／mL，转化生长因子一81（TGF-131）20ng／mL的RPMI1640培养基培养7d，LPS刺激24h使其成熟（所有实验动物处置符合动物伦理学标准）。显微镜下观察DC的形态、超微结构，流式细胞仪检测其免疫表型，CCK一8法检测刺激淋巴细胞增殖活性，趋化实验检测细胞迁移能力。结果①利用不同细胞因子组合的培养体系能诱导生成具有典型树枝状突起的DC细胞，透射电镜下观察到mDC放射状突起多而密集，细胞表面褶皱多，imDC、DCreg放射状突起较稀疏，细胞表面褶皱减少；②mDC高表达CD80、CD86、CDllc和IA／IE分子，imDC低表达CD80、CD86、IA／IE分子，高表达cDllc，DCreg高表达CD45RB，低表达CDllc、CD80、CD86；③DCreg组和imDC组体外刺激异基因淋巴细胞增殖能力较弱。3组DC刺激异基因淋巴细胞增殖率比较，差异有统计学意义（F-163．24，P〈O．01）；④DCreg的趋化迁移能力最强。3组迁移率相比，差异有统计学意义（F-560．644，P〈0．01）。结论应用细胞因子GM—CSF，IL-10和TOF-能成功诱导培养出小鼠骨髓源性CDllcCD45RB“曲调节性DC，其功能特征为移植免疫耐受的进一步研究奠定实验基础。Objective To induce the mouse bone marrow-derived CD11cI^wCD45RBhigh regulatory dendritic cells （DCreg） and identify them from the aspects of cell morphology, immunological phenotype, stimulating T lymphocytes proliferation and the ability of chemotaxis in vitro. Methods Mononuclear cells obtained from C57BL/6 mice bone marrow cells were seeded in six well plates with RPMI 1640 culture medium at a density of 1 X 106 cells/well. The culture medium was added different cytokines according to the group, which made the ceils differentiate to different types of DC. The cells were divided into three groups： Olmmature DC （imDC） group were cultured for 7 d in RPMI 1640 culture medium containing recombinant murine granulocyte macrophage-colony stimulating factor （rmGM-CSF） 20 ng/mL, recombinant murine inerleukin-4 （rmIL-4） 10 ng/mL. Mature DC （mDC） group were cultured for 6 d in RPMI 1640 culture medium containing rmGM-CSF 20 ng/mL,IL-4 10 ng/mL, then stimulated with lipopolysaccharide （LPS）for 24 h. （~ DCreg group were cultured for 7 d in RPMI 1640 culture medium containing rmGM-CSF 20 ng/ mL, IL-10 20 ng/mL and transforming growth factor-Ill （TGF-131） 20 ng/mL, then stimulated with LPS for 24 h. All experiments were carried out in accordance with relevant regulatory standards for animal ethics. We identified the morphological phenotype and ultrastrueture of DC by optical microscope and transmission electron microscope （TEM）, the immunological phenotype by flow cytometry （FCM） and the ability of migration by chemotactic assy. At 5 d in the mixed lymphocyte reaction,the proliferative capacity of T cells stimulated by different types of DC were examined by the cell count of kit-8 （CCK-8） and the ability of migration by chemotactic assy. Results OUnder transmission electron microscope, radial protrusions and folds on the surface of mDC was more and dense compared with imDC and DCreg. ~） The result of immunological phenotype detected by FCM showed that CD80, CD86,CDllc and IA/IE w

关 键 词：树突状细胞 调节性 免疫耐受 小鼠 细胞因子 细胞培养 

分 类 号：R392[医药卫生—免疫学]