人外周血来源树突状细胞的诱导及培养方法的研究

Study on induction and identification of dendritic cells from human peripheral blood

目的探讨从人外周血中大量培养树突状细胞(DC)的方法,并在表型、形态、结构等方面进行鉴定。方法采用5步法从人外周血中大量培养DC:①Ficoll不连续密度梯度离心分离外周血单个核细胞(PBMC);②RPMI-1640培养基离心洗涤去除血小板;③利用DC前体细胞的短暂粘附性去除悬浮细胞及粘附性较强的细胞;④用重组人白介素4(rhIL-4)、重组人粒-巨噬细胞集落刺激因子(rhGM-CSF)自外周血单个核细胞诱导分离DC;⑤用重组人肿瘤坏死因子诱导产生成熟的DC。用荧光显微镜检测DC表面分子的表达,在扫描电镜下观察DC的形态。结果获得了具有典型特征的DC前体细胞,在GM-CSF和rhIL-4协同诱导9 d后,荧光显微镜下可见DC表面CD83、CD40、HLA-DR的表达。扫描电镜观察可见:DC细胞形态不规则,从胞体辐射状伸出数个粗细不等的突起,有的长度可达胞体的2～3倍,长突起存在分叉及汇合现象。在长突起的基部存在大量短小的圆形或椭圆形突起。结论人外周血来源DC的5步培养法可获得较高数量的典型DC,且该法稳定、简便、经济,为DC的临床免疫治疗提供了实验依据。

Objective To discuss and establish the method for mass culturing dendritic cells from human peripherial blood.Methods The following five steps for mass culturing dendritic cells from human peripherial blood are established：①To use Ficoll discontinuous density gradient centrifugation to separate PBMC;②To use RPMI 1640 culture medium for centrifugalization and removing thrombocytes;③To use the ephemeral adhesion of DC precursors to remove suspending cells and strongly adhesive cells;④DC were isolated from human peripheral blood with the induction of mononuelear cells by recombinant human granulocyte/macrophage colony stimulating factor and recombinant human interleukin 4.⑤Mature dendritic cells were induced by recombinant human tumor necrosis factor.The expression of surface molecules of DC was examined by fluorescent microscopy and scanning electron microscopy for morphological study.Results Nine days after induction,the expression of CD40,HLA-DR and CD80 had been observed under fluorescent microscopy.The morphological appearance of DC was irregular under SEM.Numerous dendrites radially protrude from cell bodies,some of them being as long as 2 or 3 times of cell body,and the dendrites cross and fuse with each other.A large quantity of circular or oval small dendrites exist at the base of long dendrites,they were in conformity with the morphology of DC as reported before.Conclusion Highly effective and typical DC were obtained by this method,and this method is stable,simple,economic,and it can be used for clinical immunotherapy.