摘要目的拟建立慢性粒细胞白血病(CML)树突状细胞(DCs)体外无血清培养体系,以期将来用于CML的过继性免疫治疗.方法小牛血清(FCS)、无血清(SFM)及自体血清分别培养CML病人外周血/骨髓CD34+细胞或单个核细胞,以SCF、GM-CSF、TNF-α和IL-4(A)与GM-CSF、TNF-α和IL-4(B)两组不同的细胞因子作对照.间接免疫荧光法及流式细胞术鉴定CML-DCs表型,甲基四噻唑蓝(MTT)法测定CML-DCs刺激同种异体T细胞反应能力,G显带技术检测培养DCs携带Ph染色体比例,MTT法评价CML-DCs刺激自身T细胞杀伤自体白血病细胞效应.结果 8例CML慢性期病例,SFM条件下A组培养体系在总细胞扩增倍数及DCs比例两方面均明显优于B组,SFM同FCS体系相比无显著差异,但自体血清培养结果低于以上两种体系.CML-DCs MHC-Ⅱ类分子呈高表达(>50%),但CD83和CD86表达比例不高(10%-50%),且刺激同种异体T细胞反应能力不强.3例患者检测了培养CML-DCs Ph染色体比例,分别为100%、98% 及60%,与培养前比例基本吻合;同时以CML-DCs同自身T细胞、自体白血病细胞共孵育,测其杀伤率为38.5%±6.5%(效靶比为40∶1).结论①建立了稳定的无血清培养CML-DCs体系;②CML-DCs MHC-Ⅱ类分子显著表达,CD83和CD86表达比例不高,刺激同种异体T细胞反应能力不强;③证实此DCs携带Ph染色体,能刺激自体T细胞杀伤自身白血病细胞.

abstractsObjective To establish a serum-free culture system of dendritic cells (DCs) from chronic myeloid leukemia (CML) cells so that DCs vaccine may be applied to the adoptive immunotherapy of CML in the near future. Methods Fetal calf serum, serum-free medium and autologous serum were used for culture of DCs. The usage of cytokines was classified into two groups: group A (stem cell factor, granulocyte/macrophage colony-stimulating-factor, tumor necrosis factor-α and interleukin-4) and group B (granulocyte/macrophage colony-stimulating-factor, tumor necrosis factor-α and interleukin-4). The phenotypes of DCs were analyzed by using indirect immunofluorescence and flow cytometry. Mixed leukocyte responses were performed by methyl thiazolyl tetrazolium (MTT) assay. Chromosome analysis of DCs can be achieved by displaying G banding. T cells from CML patients were stimulated with autologous DCs and T-cell cytotoxicity was measured by (MTT) assay. Results CD34+ cells or mononuclear cells were obtained from peripheral blood or bone marrow samples of eight patients of chronic-phase CML. Group A of serum-free medium was better than group B in expansion of total cell numbers and the rate of DCs. These results of serum-free medium were not significantly different from those of fetal calf serum medium, but the results of autologous serum medium were inferior to two groups above. The expression of major histocompatibility complex class Ⅱ antigen on the surface of DCs was notable (＞50%), but the expression of CD83 and the costimulatory molecules CD86 was not noticeable (10%-50%). Although CD1a+/CD14 DCs were potent stimulators of allogeneic lymphocytes, expansion of T cells from normal volunteers were not significant (average 27.2 fold at DCs∶T cells ratio of 1∶10). At day 12, CD1a+ cells from three patients were studied by displaying G banding and Ph+ cells in these populations were 100%, 98% and 60%, respectively. At an effector∶target ratio of 40∶1, 32% to 45% cytotoxicity was noted with DC-stimulated T cells against autologous leukemia cells. Conclusions A stable serum-free culture system of CML-DCs was established. The expression of CD83 and CD86 on the surface of CML-DCs and DCs' potent stimulation of allogeneic lymphocytes were not notable. DCs in CML patients can be derived from the malignant clone and these malignant DCs could induce anti-leukemic reactivity in autologous T lymphocytes without the necessity for additional exogenous antigens.