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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 dendritic cells (DCs) from human bone marrow hematopoietic progenitor cells in vitro and measure their phenotype and T cell stimulating activity.Methods: Mini-MACS technique was used to detect DCs from normal human bone marrow, cord blood CD34 ~ + hematopoietic stem cells were isolated and cultured in vitro for 2 weeks with recombinant hGM-CSF, hTNF-α and hIL-3. Flow cytometry was used to detect the phenotype of the expanded cells and the expression of IL-12 in cells. Mixed lymphocyte reaction was used to detect the T cell stimulating activity of DCs.Results: The high purity (> 90%) CD34 + hematopoietic stem cells were isolated from normal human bone marrow and umbilical cord blood, and the recombinant hGM-CSF and hTNF- The results of FACS showed that the amplified DCs expressed HLA-DR, CD40, CD54, CD80 and CD86 molecules, and the cells with hIL-12 P35 , P40 subunit expression.Compared with DCs cultured in peripheral blood mononuclear cells, DCs expanded by CD34 + stem cells had more ability to stimulate allogeneic T cell proliferation.Conclusion: Human CD34 + stem cells in vitro After induction culture, can produce a large number of functionally-mature DCs, so as to further develop the basic and clinical research of DCs The foundation.