CAJ | 학술논문

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.
목적 탐토우화인γhG-CSF동원적외주혈채집물래원적수돌상세포(DC)성숙유도조건.방법 이용밀도제도리심법종중조인립세포집락자격인자(γhG-CSF)동원적외주혈채집물분리출단개핵세포,이중조인립세포-거서세포집락자격인자(GM-CSF)화중조인백세포개소-4(IL-4)체외유도성불성숙수돌상세포(imDC),분별채용전통세포인자계미주(IL-1β、IL-6、TNF-α、PGE2)화개량세포인자계미주(IL-1β、IL-6、TNF-α、polyI:C、CpG ODN)자격성숙.3d후수획성숙DC,관찰세포형태、류식세포의검측DC적내탄능력통과이류청산형광소표기적포취당(FITC-Dextran)、검측DC표면CD83、CD1a、CD80、HLA-DR、CD86화CCR-7,매련면역흡부법(ELISA)검측기IL-12적분비,MTT법검측기자격림파세포증식활성.결과 량충방법균능유도DC성숙,이개량세포인자계미주효과경가,FITC-Dex-tran적내탄능력명현하강；CD83、CD1a적표체솔분별시84.44％、87.94％ (P ＜0.05)；IL-12분비량명현증고,성숙DC능유효적자격T림파세포증식.결론 개량세포인자계미주법가능시유도DC성숙적최가방법.
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.