非贴壁法体外诱导人外周血单核细胞生成树突状细胞的研究

Generation of mature dendritic cells from peripheral blood monocytes not purified from the PBMCs by calcium ionophore A23187 and GM-CSF in vitro

目的探讨新型诱导剂钙离子载体A23187联合重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)通过非贴壁法体外诱导人外周血单核细胞(monocytes,Mo)生成树突状细胞(dendritic cell,DC)的可行性。方法采用密度梯度离心法分离出人外周血单个核细胞(peripheral blood mononuclearc ell,PBMC),一组通过贴壁法分离出Mo,再加入rhGM-CSF+A23187,称贴壁法组。另一组直接加入rhGM-CSF+A23187,称非贴壁法组。通过光镜观察细胞形态的变化,流式细胞仪检测DC细胞的表面标志。结果两组诱导培养的DCs都具有典型的树突形态;与贴壁法比较,DC表面分子CD14-CD83+(39.2%vs40.9%)、CD14-CD1a+(19.6%vs18.3%)、CD14-CD86+(47.1%vs46.0%)、CD14-CD40+(30.5%vs32.8%)的表达无明显差异,P值均大于0.05。结论新型诱导剂钙离子载体A23187联合rhGM-CSF通过非贴壁法能有效地诱导Mo生成成熟的DCs。

Objection The aim of this study was to evaluate the feasibility of calcium ionophore （CI） A23187 and human recombinant granulocyte-macrophage colony stimulating factor （rhGM-CSF） on the cul- tivation of dendritic cell （DC） from healthy human peripheral blood monocytes （Mo） not purified from the PBMC in vitro. Methods PBMC isolated from peripheral blood of healthy human by Ficoll-Hypaque density gradient separation were separated into two groups. The monocytes purified through adhensiveness from the cells in group A were cultured in the presence of rhGM-CSF and calcium ionophore （CI） A23187. The cells in group B were cultured with additional rhGM-CSF and CI A23187 directly. The cells in both groups were loaded by freeze-thawed K562 cells on the first day and were harvested in 96 hours of cultivation. The mor- phology of the DCs was observed by microscope all through the process. The surface antigens of the induced cells after culturing for 96 hours were analyzed by fluorescence-activated cell sorting （FACS）. Results Typi- cal morphological features of DCs could be observed in both groups. On 96 hours, the expression percentage of DCs in both group were as follows： CD14-CD83＋（39.2% vs 40.9%）, CD14-CD1a＋（19.6% vs 18.3%）, CD14- CD86＋（47.1% vs 46.0%）, andCD14-CD40＋（30.5% vs 32.8%）, and there was no significant difference in both group（P 〉 0.05）. Conclusion CI A23187 combined rhGM-CSF could effectively induce peripheral blood Mo without purification from the PBMC into DC.