人外周血来源的成熟树突状细胞的体外无血清培养

目的探讨无血清条件下健康人外周血单个核细胞（PBMC）快速诱导生成成熟树突状细胞（dendritic cell,DC）的体外培养方法。方法分离获取健康者外周血PBMC,将细胞分为血清组和无血清组：设血清组A组为对照组,B、C、D组为无血清组。A组加入10%FCS的RPMI1640完全培养液,B组加入单核/巨噬细胞无血清培养液MΦ-SFM,两组均加入rhGM-CSF＋IL-4＋rhTNF-α培养9d;C组加入钙离子载体（CI）A23187,D组加入rhGM-CSF＋A23187,两组细胞均培养于MΦ-SFM中48h。于倒置显微镜下观察细胞形态,流式细胞术分析细胞表型,MTT比色法检测各组细胞刺激同种异体T淋巴细胞增殖的能力。结果在无血清条件下,单独给予A23187或A23187＋rhGM-CSF联合刺激48h,均可诱导PBMC分化成具有典型形态的DC;与血清组细胞相比,细胞表面标志CD80、CD86和CD83分子具有相似的高表达（P〉0.05）,对同种异体T淋巴细胞的刺激能力无显著性差异（P〉0.05）。结论在无血清培养条件下配合使用钙离子载体A23187,可以更快速、有效地诱导健康人PBMC分化成更成熟、功能更强的DC。

Culture of mature dendritic cells from human peripheral blood monocytes with serum-free medium in vitro

Objective To explore the method of culture of mature dendritic cells（DCs） induced from human peripheral blood monocytes（PBMCs） with serum-free medium in vitro.Methods PBMCs isolated from healthy donors were separated into serum group and serum-free group.Group A were cultured in serum medium as control group.Group B,C and D were cultured in serum-free medium.Group A were cultured with rhGM-CSF＋IL-4＋rhTNF-α in 10% FCS complete RPMI medium1640 for 9 days.Group B were grown with the same cytokine combination as Group A in macrophage serum-free medium（M？-SFM）.Group C were added calcium ionophore（CI） A23187 and Group D were added A23187＋rhGM-CSF.Both groups were grown in M？-SFM for 48h.The morphology of the induced cells was observed by inverted phase contrast microscope,and the phenotypes were analyzed by flow cytometry.MTT colorimetry was used to detect the proliferation of allogeneic T cells.Results PBMCs treated with A23187 or A23187＋rhGM-CSF in serum-free medium presented the typical morphology of DCs.Comparing with the serum group,the cells in these two groups not only exhibited similar high expression of surface markers CD80,CD86 and CD83（P0.05）but also had strong allo-stimulatory capability（P0.05）.Conclusions Under the serum-free condition,the mature and functional DCs could generate rapidly and efficiently from human PBMCs treated with calcium ionophore A23187.