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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。
Objective To investigate the in vitro culture method of dendritic cells (DCs) induced by peripheral blood mononuclear cells (PBMCs) from healthy people under serum-free conditions. Methods Peripheral blood PBMCs from healthy individuals were isolated and divided into serum group and serum-free group. Serum group A as control group, B, C, D group as serum-free group. Group A was supplemented with 10% FCS in RPMI1640 complete medium, Group B was added monocyte / macrophage serum-free medium MΦ-SFM, both groups were added rhGM-CSF + IL-4 + rhTNF- Calcium ionophore (CI) A23187, group D rhGM-CSF + A23187 was added, and both groups were cultured in MΦ-SFM for 48h. Cell morphology was observed under an inverted microscope. Cell phenotypes were analyzed by flow cytometry. The proliferation of allogeneic T lymphocytes was measured by MTT assay. Results Under the condition of no serum, the combination of A23187 or A23187 + rhGM-CSF for 48h could induce PBMC to differentiate into typical DCs. Compared with the serum group cells, the cell surface markers CD80, CD86 and CD83 were similar (P> 0.05). There was no significant difference in the ability to stimulate allogeneic T lymphocytes (P> 0.05). Conclusion Combined use of calcium ionophore A23187 under serum-free culture conditions can induce healthy human PBMC to differentiate into more mature and more powerful DC more quickly and effectively.