人γhG-CSF动员的外周血采集物来源的树突状细胞成熟诱导的研究
The study of maturation induction of γhG-CSF mobilized peripheral blood grafts-derived dendritic cells
目的 探讨优化人γhG-CSF动员的外周血采集物来源的树突状细胞(DC)成熟诱导条件.方法 利用密度梯度离心法从重组人粒细胞集落刺激因子(γhG-CSF)动员的外周血采集物分离出单个核细胞,以重组人粒细胞-巨噬细胞集落刺激因子(GM-CSF)和重组人白细胞介素-4(IL-4)体外诱导成不成熟树突状细胞(imDC),分别采用传统细胞因子鸡尾酒(IL-1β、IL-6、TNF-α、PGE2)和改良细胞因子鸡尾酒(IL-1β、IL-6、TNF-α、polyI:C、CpG ODN)刺激成熟.3d后收获成熟DC,观察细胞形态、流式细胞仪检测DC的内吞能力通过异硫氰酸荧光素标记的葡聚糖(FITC-Dextran)、检测DC表面CD83、CD1a、CD80、HLA-DR、CD86和CCR-7,酶联免疫吸附法(ELISA)检测其IL-12的分泌,MTT法检测其刺激淋巴细胞增殖活性.结果 两种方法均能诱导DC成熟,以改良细胞因子鸡尾酒效果更佳,FITC-Dex-tran的内吞能力明显下降;CD83、CD1a的表达率分别是84.44%、87.94% (P <0.05);IL-12分泌量明显增高,成熟DC能有效的刺激T淋巴细胞增殖.结论 改良细胞因子鸡尾酒法可能是诱导DC成熟的最佳方法.
更多Objective To investigate the most effective strategy for mature induction of γh G-CSF mobilized peripheral blood grafts-derived dendritic cells.Methods The peripheral blood mononuclear cells (PBMCs) were isolated from γh G-CSF mobilized peripheral blood grafts,by using density gradient centrifugation method,then imDC was induced in the presence of cytokines GM-CSF and IL-4.The immature DC was pulsed with each of a traditional cocktail of cytokines (TNF-α、IL-β 、IL-6 、PGE2) or a modified cocktail of cytokines (IL-1 β 、IL-6 、TNF-α、polyI:C 、CpG ODN).DC was harvested after 3d induction,Cells morphology was Observed,FITC-Dextran endocytic activity was measured by FCM.Detected the surface markers for DC maturation CD83,CD1a,CD80,HLA-DR,CD86 and CCR-7 by FCM.IL-12 production was detected by ELISA.The capacity of DC for T cell activation was detected by MTT assay.Results The two means all induce DC mature.The most effective method for induction of maturation was modified cocktail of cytokines.FITC-Dextran endocytic activity was clearly declined.the expression rate of CD83 and CD1a were up to 84.44% and 87.94% (P <0.05).The amount of IL-12 production increased significantly,mature DC can effective stimulation T lympho cyte proliferation.Conclusion The most effective method for inducing DC' maturation was modified cocktail of cytokines.
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