FVB小鼠骨髓源树突状细胞的培养

Culturing of dendritic cells from bone marrow of FVB mice

目的建立FVB小鼠骨髓源树突状细胞体外培养和扩增方法 ,观察其形态和特征。方法在无菌条件下提取FVB鼠骨髓细胞,分离单个核细胞,用IL-4和GM-CSF联合协同诱导下培养,加用磷酸脂多糖刺激。应用光镜下观察树突状细胞的形态,流色细胞仪检测鉴定其生物学特征。结果联合培养小鼠骨髓细胞3 d后,可见细胞形态发生改变,细胞形态不规则。培养第6～8 d,光镜下显示,细胞表面出现较多毛刺样突起,拉长,细胞表面不规则,呈树突状突起,为典型的DC特征。流式细胞鉴定为髓系树突状细胞,单纯树突状细胞组的细胞中度表达MHCⅡ类分子,低水平表达共刺激分子,刺激T细胞增殖的能力较弱磷酸酯多糖-树突状细胞组细胞高水平表达MHCⅡ类分子和共刺激分子。结论采用FVB小鼠骨髓源树突状细胞应用IL-4联合GM-CSF培养FVB小鼠骨髓细胞,可大量扩增成熟DC,培养的DC符合其自身的特性。

Objective To establish a method for cultivation and amplification of mouse bone marrow-derived dendritic cells in vitro,and to observe cell morphology and related characteristics.Methods Under aseptic condition,bone marrow cells were extracted from the tibia and femur bones of FVB mice.Bone marrow mononuclear cells were isolated,and cultured in the RPMI 1640 medium with recombinant mouse granulocyte-macrophage colony-stimulating factor（rmGM-CSF） and interleukin（IL）-4 in vitro.Dendritic cells obtained were divided into two groups： lipopolysaccharide（LPS）-dendritic cell and sample dendritic cell.Dendritic cells in the LPS-dendritic cell group were treated with LPS for stimulating and the dendritic cells in the simple dendritic cell group were used as controls without treatment.The morphology of the dendritic cells was observed by inverted microscope,The biological characteristics of the dendritic cells were identified by flow cytometry.Results Two weeks after culture,the cultured cells displayed the typical morphology of dendritic cells.Under the inverted microscope,the surface of the cultured cells was irregular,and there were many dendrite-like processes on the surface.The cultured cells were indentified as myeloid dendritic cells by flow cytometry.High expressions of MHC-Ⅱ,CD80,CD86 and CD11c were observed on the surface of the cultured cell.In the simple dendritic cell group,MHC-Ⅱ expression was at a moderate level,CD80,CD86 and CD11c expressions were at a low level,and the cultured cells had the weak ability to stimulate the proliferation of T cells.In the LPS-dendritic cells group,MHC-II,CD80,CD86 and CD11c expressions were at a high level.Conclusion The method of primary culture used in this experiment can produce a large amount of dendritic cells in vitro,and the cultured cells displays the typical morphology of dendritic cells with a higher purity.