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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 DCsMHC Ⅱ类分子呈高表达(>5 0 % ) ,但CD83和CD86表达比例不高 (10 % - 5 0 % ) ,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 DCsPh染色体比例 ,分别为 10 0 %、98%及 6 0 % ,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 4 0∶1)。结论 ①建立了稳定的无血清培养CML DCs体系 ;②CML DCsMHC Ⅱ类分子显著表达 ,CD83和CD86表达比例不高 ,
Objective To establish a serum-free culture system for chronic myeloid leukemia (CML) dendritic cells (DCs) in vitro for future adoptive immunotherapy of CML. Methods Peripheral blood/bone marrow CD34+ cells or mononuclear cells were cultured in calf serum (FCS), serum-free serum (SFM) and autologous serum. The SCF, GM CSF, TNF α and IL 4 (A) and GM CSF, TNF Two different cytokines, α and IL 4 (B), were used as controls. Indirect immunofluorescence and flow cytometry were used to identify the CML DCs phenotype, MTT assay was used to measure the ability of CML DCs to stimulate allogeneic T cells, G banding technique was used to detect DCs carrying Ph chromosomes ratio, and MTT assay. CML DCs were evaluated for their ability to stimulate autologous T-cell killer autologous leukemia cells. Results In the 8 chronic cases of CML, under the condition of SFM, the proliferation rate of group A and the proportion of DCs in group A were significantly better than that of group B. There was no significant difference between SFM and FCS system, but the results of self-cultured serum were lower. The above two systems. CML DCsMHC class II molecules were highly expressed (>50%), but the ratio of CD83 and CD86 expression was not high (10% - 50%), and the ability to stimulate allogeneic T cells was not strong. The percentage of chromosomes in cultured CML DCsPh was detected in 3 patients, which were 100%, 98%, and 60%, respectively, which were basically consistent with the ratios before culture. At the same time, CML DCs were co-incubated with autologous T cells and autologous leukemia cells to determine the killing rate. It is 38.5 % ± 65% (effect ratio: 4:1). Conclusions 1 Stable serum-free cultured CML DCs system was established; 2CML DCsMHC II molecules were significantly expressed, and CD83 and CD86 expression ratios were not high.