人CD4^+CD25^+调节性T细胞的体外培养扩增  

Isolation and amplification of human CD4^+CD25^+ regulatory T cells in vitro

作　　者：孙磊[1] 吴静静 易寿南 陈丽[1] 

机构地区：[1]山东大学齐鲁医院内分泌科,济南250012 [2]悉尼大学westmead医院CTRR

出　　处：《山东大学学报（医学版）》2010年第12期1-4,10,共5页Journal of Shandong University:Health Sciences

摘　　要：目的建立人CD4+CD25+Treg细胞的分离和体外扩增方法,并检测扩增细胞的表型及体外免疫抑制功能。方法免疫磁性分离(MACS)方法分离人CD4+CD25+Treg细胞,加入刺激因子与之共培养扩增,用台盼蓝染色法和流式细胞法检测细胞的活性和纯度,流式细胞法检测细胞主要表型标记,实时定量PCR检测Treg核心标记Foxp3的表达,混合淋巴细胞反应试验分析其免疫抑制功能,ELISPOT方法检测产生细胞因子的细胞数目。结果扩增2～3周的CD4+CD25+Treg细胞数目达到新鲜细胞的3500倍,活性>97%,纯度>96%;扩增的细胞与新鲜的细胞保持了相似的表型、Foxp3表达,其抑制功能略强于新鲜的CD4+CD25+Treg细胞,其差异具有统计学意义(71%vs51%,P<0.05),可能与其产生抑制性细胞因子的细胞数目增加有关。结论本实验室创建的分离和体外扩增人CD4+CD25+Treg细胞的方法,可以大量扩增出高纯度、有活力且不影响其抑制功能的Treg细胞,解决了CD4+CD25+Treg细胞数目少的问题。Objective To establish a method for isolating and expanding human CD4＋CD25＋ regulatory T（Treg）cells in vitro,and to identify the purity,phenotype,activity and function to suppress xenogeneic immune responses of fresh and expanded Treg cells.Methods Human Treg cells were isolated by magnetic activated cell sorting（MACS）and expanded by CD3/CD28 expansion beads,IL-2 and rapamycin.Activities and the purity of the fresh and expanded cells were detected by trypan blue staining and FACS,respectively.The phenotype and Foxp3 gene expression were analyzed by FACS and real-time PCR,respectively.The suppression of fresh or expanded human Treg cells was detected by the mixed lymphocyte reaction（MLR）assay.Cytokine-producing cells in coculture were examined by ELISPOT.Results Human CD4＋CD25＋ Treg cells were expanded up to 3500-fold the number of fresh Treg cells after 2-3 weeks of culture.The activity of the expanded CD4＋CD25＋ Treg cells was 〉97 %,and the purity of them was 〉96 %.The expanded Treg cells had a similar phenotype and Foxp3 expression to fresh Treg cells.In the coculture experiment（MLR assay）,expanded Treg cells were more potent at suppressing CD4＋CD25-T cell-mediated anti-porcine xenogeneic responses compared with fresh Treg cells,and the difference was significant（71% vs 51%,P〈0.05）.The enhanced suppression of expanded Treg cells was associated with increased secretions of IL-4 and IL-10.Conclusions The method established in the study for isolation and expansion of human CD4＋CD25＋ Treg cells is effective to obtain sufficient cells,which have high purity and activity,and the suppressive function is uninfluenced.

关 键 词：人CD4^+CD25^+T细胞 体外扩增 表型标记 免疫抑制 细胞因子 

分 类 号：R392.33[医药卫生—免疫学]