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目的 探讨人脐血CD+34 细胞体外诱导树突状细胞 (dendriticcells ,DCs)的可能性 ,并检测DCs的功能。方法 利用吸附单克隆抗体 磁珠分离系统 (MACS)获得脐血CD+34 细胞 ,体外以重组的人干细胞集落刺激因子 (hSCF)、人粒 巨噬细胞集落刺激因子 (hGM CSF)、人肿瘤坏死因子α(hTNF α)、人酪氨酸激酶受体家族III的配体 (hFL)诱生DCs。流式细胞仪检测DCs的表型 ,3H TdR掺入法检测DCs体外刺激同种异体T细胞增殖的功能 ,最后通过乳酸脱氢酶法检测DCs活化的T细胞对肿瘤细胞的杀伤性。结果 从人脐血分离到的CD+34 细胞 ,经hSCF、hGM CSF、hTNF α、hFL共同培养后 ,第 7天镜下即可见典型形态的DCs ,第 10天时明显增多 ,细胞表型检测见CD+1a 细胞的比例增加到 (2 8± 4) %(P <0 0 1) ,人类白细胞抗原DR(HLA DR)表达占 (94± 11) %。在三种不同的混和比例下 ,DCs均可刺激异源T细胞增殖 ,其中以DCs∶T细胞为 1∶5时增殖最明显。而且被激活的T细胞对三种不同组织来源的肿瘤细胞均产生了明显的杀伤性 (P <0 0 1)。结论 人脐血CD+34 细胞体外经细胞因子诱导培养 ,可生成大量具有典型功能的成熟DCs。
Objective To investigate the possibility of dendritic cells (DCs) induced by cord blood CD + 34 cells in vitro and the function of DCs. Methods Umbilical cord blood CD34 + cells were obtained by MACS. Recombinant human stem cell colony stimulating factor (hSCF), human granulocyte macrophage colony stimulating factor (hGM CSF), human tumor necrosis Factor α (hTNF α), a ligand of human tyrosine kinase receptor family III (hFL), induces DCs. The phenotypes of DCs were detected by flow cytometry. 3H TdR incorporation method was used to detect the proliferation of allogeneic T cells stimulated by DCs in vitro. Finally, the cytotoxicity of DCs activated T cells to tumor cells was detected by lactate dehydrogenase assay. Results CD34 cells isolated from human umbilical cord blood were co-cultured with hSCF, hGM CSF, hTNF α, and hFL. The typical morphology of DCs was found on the 7th day. The number of DCs increased on the 10th day. The proportion of CD + 1a cells increased to (28 ± 4)% (P <0.01), and HLA DR expression accounted for 94 ± 11%. DCs stimulated the proliferation of allogeneic T cells under three different mixing ratios, and the most obvious proliferation was DCs: T cells at 1: 5. In addition, activated T cells produced significant cytotoxicity on tumor cells derived from three different tissues (P <0.01). Conclusion Human umbilical cord blood CD34 cells cultured in vitro induced by cytokines can produce a large number of mature DCs with typical functions.