摘要
目的:体外诱导、培养人外周血单个核细胞获得不同成熟阶段的树突状细胞(DCs)及其功能鉴定,为肿瘤免疫治疗提供基础。方法:密度梯度离心法分离人外周血单个核细胞,黏附6小时后祛除悬浮细胞,第1阶段加入含重组人粒细胞-巨噬细胞集落刺激因子和重组人白细胞介素-4的RPMI1640培养液诱导培养7天,获得未成熟DCs;第2阶段在重组人粒细胞-巨噬细胞集落刺激因子和TNF-α联合诱导下培养至14天,获得成熟的DCs。对DCs的形态进行显微镜下观察,用流式细胞仪检测其表型,用混合淋巴细胞反应检测其刺激T细胞增殖能力。结果:获得的未成熟DCs中度表达CD1a、共刺激分子,高表达HLA-DR分子,在DCs的成熟期共刺激分子、HLA-DR及CD83、CD11c分子均高度表达,刺激同种异型T淋巴细胞增殖的能力强。结论:成功获得了诱导人外周血单个核细胞来源的大量纯度较高的DCs并验证了其抗原呈递能力。
Objective：To induce and culture purity dendritic cells（DCs） of different maturation phase from human peripheral blood monocytes in vitro.Methods：The peripheral blood mononuclear cells were isolated from human peripheral blood with density gradient centrifugation.The first step was to work on a 7-day culture of monocytes in medium supplement with 100ng/ml rhGM-CSF and 5ng/ml rhIL-4,and the cells were actually immature.In the second step,the cells were cultured in the medium supplement with GM-CSF and TNF-α,and cultured until the 14th day.Mature DCs were obtained.Cells harvested were identified for their morphology by microscope,properties of DCs detected by FCM,function with MLR method.Results：Immature dendritic cells expressed CD1a molecules and costimulating molecules in middle level,HLA-DR in high level.In maturation phase DCs expressed costimulating molecules,HLA-DR molecules,CD83,and CD11c highly.These DCs could stimulate proliferation of allogenic T lymphocytes.Conclusion：The method to obtain DCs of different maturation phases from human peripheral blood monocytes is successfully set up.A number of DCs with high purity are obtained.
