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目的建立来源于外周血的树突状细胞(DCs)的纯化培养方法并观察DCs的形态及功能。方法以Fi-coll密度梯度离心法从正常人外周血中分离出外周血单个核细胞(PBMNC)后,用体外培养的手段,采用培养黏附法或经免疫磁珠筛选法,获得单核细胞;分别加入不同浓度的细胞因子(重组人粒细胞-巨噬细胞集落刺激因子重组人白介素4重组人肿瘤坏死因子α)诱导培养,培养12 d诱导出DCs;用光镜和电镜观察培养的DCs,用流式细胞仪检测DCs表面的细胞表型(CD80、CD83、CD86、CD1α、HLA-DR),MTT法检测DC对T细胞的刺激增殖效应。结果在体外诱导出外周血来源的成熟DCs,电镜和光镜分析表明具有DCs的典型形态,所培养的细胞表面高表达CD80、CD83、CD86、CD1α、HLA-DR,并具有刺激异基因淋巴细胞增殖的能力。结论应用外周血来源的单个核细胞,采用培养黏附法或免疫磁珠分选法分选出的单核细胞,细胞因子培养12 d可得到成熟正常的DCs。
Objective To establish a method for the purification and culture of dendritic cells (DCs) derived from peripheral blood and to observe the morphology and function of DCs. Methods Peripheral blood mononuclear cells (PBMNC) were isolated from peripheral blood of healthy people by Fi-coll density gradient centrifugation. Monocytes were obtained by culturing in vitro or by culture with magnetic beads. DCs were induced by different concentrations of recombinant human interleukin-4 (recombinant human TNF-α) and cultured for 12 days respectively. DCs were cultured under light microscope and electron microscope. The cell phenotypes (CD80, CD83, CD86, CD1α, HLA-DR) on the surface of DCs were detected by flow cytometry. The proliferation of T cells stimulated by DC was detected by MTT assay. Results The mature DCs derived from peripheral blood were induced in vitro. Electron microscopy and light microscopy showed typical DCs. The cultured cells expressed high levels of CD80, CD83, CD86, CD1α and HLA-DR on the surface of cells and stimulated allogeneic lymphocyte proliferation Ability. Conclusions Mononuclear cells derived from peripheral blood can be used to culture monocytes isolated by culturing adhesion or immunomagnetic beads sorting method. After cultured for 12 days, mature and normal DCs can be obtained.