人骨髓、脐血CD34^+干细胞体外扩增的树突状细胞的表型及其T细胞刺激活性分析

Phenotype and T Cell Stimulating Activity of Dendritic Cells Expanded from Human Bone Marrow and Cord Blood CD34^(+) Hematopoietic Progenitor Cells

目的:从人骨髓造血前体细胞体外培养扩增树突状细胞(dendritic cells,DCs),测定其表型及T细胞刺激活性.方法:采用Mini-MACS分离技术,从正常人骨髓、脐血分离CD34^+造血干细胞,体外以重组hGM-CSF,hTNF-α,hIL-3诱导培养2周,流式细胞术检测扩增细胞的表面表型及细胞内IL-12的表达,体外同种混合淋巴细胞反应检测扩增DCs的T细胞刺激活性.结果:从正常人骨髓、脐血分离得到高纯度(>90%)的CD34^+造血干细胞,经重组hGM-CSF,hTNF-α的共同诱导培养,扩增得到大量DCs,加人hIL-3可以进一步增加DCs产量;FACS检测表明,扩增的DCs表达HLA-DR,CD40,CD54,CD80,CD86分子,细胞内有hIL-12的P35,P40亚基的表达;与外周血单核细胞培养生成的DCs相比,由CD34^+干细胞扩增的DCs具有更强的激发同种T细胞增殖的能力.结论:人CD34^+干细胞体外经诱导培养,可以生成大量功能成熟的DCs,从而为进一步开展DCs的基础及临床研究打下了基础.

Objective: To expand dendritic cells from human bone marrow and cord blood CD34+ hematopoietic progenitor cells in vitro and analyze their cell surface phenotype and T cell stimulating activity. Methods: CD34+ hematopoietic stem cells were isolated from normal bone marrow or cord blood by mini-MACS and cultured in GM-CSF, TNF-α, rhlL-3 for 2 weeks. Then the phenotype and IL-12 expression of the expanded cells were analysed by FACS. T cell stimulating activity was determined by allo-MLR. Results: Purity of human CD34+ hematopoietic stem cells isolated from normal bone marrow and cord blood was more then 90% . Large amount of DCs could be obtained by culture in presence of hGM-CSF and hTNF-α, More DCs could be obtained when hIL-3 was also added. DCs expressed HLA-DR, CD40, CD54, CD80, CD86. Intracellular cytokine assay by FACS showed that DCs expressed p35 and p40 subunits of IL-12. The proliferation of allo-T cells stimulated by CD34+ stem cell-derived DCs was more potent than that stimulated by peripheral monocyte-derived DCs. Conclusion: Large amount of mature DCs could be generated by in vitro culture of human CD34+ stem cells.