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目的 建立从人外周血单核细胞体外诱导培养成熟和激活的树突状细胞 (dendritic cell,DC)的方法。方法 从健康成人外周血分离单核细胞 (PBM) ,加入粒单系集落刺激因子 (GM-CSF) 10 0 μg/L+重组白细胞介素 -4 (rh IL -4 ) 5 0 0 k U /L体外培养 14 d,并于培养结束前 1d加入或不加入肿瘤坏死因子α (TNF -α ) (10 0μg/L ) ,流式细胞仪测定树突状细胞 (DC)的主要组织相容性复合体 (MHC) - 类分子、粘附分子和协同刺激分子 ,分析其成熟度和激活度。结果 PBM经 GM-CSF+ rh IL-4诱导培养 14 d后 ,细胞成簇 ,表型为 CD83 2 2 .6%、CD865 5 .5 %、CD11c 3 6.1%、CD64 3 .2 %、人类白细胞抗原 (HLA) -DR 13 .4% ;培养结束前 1d加入 TNF -α诱导后 ,细胞表型为 CD83 81.5 %、CD8699.3 %、CD11c 98.8%、CD64 3 .4%、HLA-DR 88.3 %。结论 GM-CSF +rh IL-4诱导 PB-M14 d,可获得大量不成熟的 DC,该体系有利于 DC扩增 ;培养结束前 1d加入 TNF-α,DC成熟度高 ,激活性好 ,适合于肿瘤免疫治疗
Objective To establish a method of culturing mature and activated dendritic cells (DCs) from human peripheral blood monocytes in vitro. Methods Mononuclear cells (PBMs) were isolated from peripheral blood of healthy adults. GM-CSF 10 0 μg / L + recombinant human interleukin-4 (rhIL-4) 500 kU / L The cells were cultured in vitro for 14 days. Tumor necrosis factor α (TNF-α) (10 0 μg / L) was added or not at 1 day before the end of culture, and the main histocompatibility complex of dendritic cells (DC) (MHC) -like molecules, adhesion molecules, and costimulatory molecules, analyzing their maturation and activation. Results PBMCs were clustered after induced by GM-CSF and rhIL-4 for 14 days. The phenotypes of PBM were CD83 22.6%, CD865.5.5%, CD11c 3 6.1%, CD643.2%, human leukocyte antigen (HLA) -DR13.4%. The cell phenotypes of CD83 were 81.5%, CD8699.3%, CD11c 98.8%, CD643.4%, and HLA-DR 88.3% after induction of TNF-α by 1 day before the end of culture. CONCLUSION: GM-CSF + rhIL-4 can induce a large number of immature DCs induced by PB-M14d, which is favorable for DC amplification. The addition of TNF-α at 1d before the end of culture induces high maturation and activation of PB, In tumor immunotherapy