钙离子载体和Poly(I:C)诱导人外周血来源的树突状细胞成熟的研究

Generation of mature dendritic cells from peripheral blood mononuclear cells by Calcium ionophore and Poly(I:C) in vitro

目的:探讨从健康人外周血中体外诱导培养出成熟树突状细胞(dendritic cell,DC)的方法。方法:用密度梯度离心法分离人外周单个核细胞(PBMC),再用贴壁法获取单核细胞后加入GM-CSF和IL-4,培养5天后分5组:第1组为对照组,仅含有上述细胞因子;第2组,加入多聚次黄嘌呤胞嘧啶核苷酸[Poly(I:C)];第3组,加入钙离子载体(A23187);第4组,加入Poly(I:C)和CI;第5组,加入TNF-α。显微镜下观察培养细胞形态,流式细胞术检测DC表型,体外同种混合淋巴细胞反应(MLR)检测DC刺激T细胞的增殖活性。结果:第四组细胞具有典型的树突状细胞的特征,且高表达CD83、CD80、CD86和CD40分子,具有更强的刺激T细胞增殖能力。结论:经过组合细胞因子诱导能够培养出PBMC来源的DC,且经CI和Poly(I:C)进一步诱导后获得更成熟和功能更强DC。

Objective：To explore the generation of mature dendritic cells （DCs） induced from human peripheral blood monocytes in vitro. Methods： Monocytes were isolated from normal human peripheral blood mononuclear cells （PBMCs）and were separated into five groups after cultured with GM-CSF and IL-4 5 days later. The control group were added only GM-CSF and IL-4. One group were added polyriboinosinic polyribocytidylic acid[Poly（I：C）], one group were added calcium ionophore（CI）,one group were added Poly （I：C） and CI, and the other group were added TNF-α The morphology of cells was observed by microscope. The surface antigens of the induced cells were analyzed by fluorescence-activated cell sorting （FACS）. T cell proliferating activity was determined by allo- MLR （mixed lymphocyte reaction） in vitro. Results：Large amount of mature DCs could be obtained from monocytes by culture in presence of GM-CSF,IL-4,Poly （I：C） and CI. DC expressed high level of CD83, CD80, CD86 and CD40, and were potent to stimulate the proliferation of allo-lymphocytes. Conclusion：The phenotypes and functions of DC generated from PBMC by the treatment of cytokine combination can be improved by calcium ionophore and Poly（I：C）.