小鼠CD4^＋CD25^＋调节性T细胞的分离纯化及鉴定  

Isolation and identification of CD4^+CD25^+ regulatory T cells

作　　者：闫峰[1] 黄振平[1] 蔡莉[2] 薛春艳[1] 丁莉莉[1] 

机构地区：[1]南京军区南京总医院眼科,江苏南京210002 [2]第四军医大学附属西京医院眼科,陕西西安710032

出　　处：《医学研究生学报》2009年第3期231-235,共5页Journal of Medical Postgraduates

基　　金：国家自然科学基金资助项目(批准号:30500550);江苏省博士后科研资助项目(批准号:0702040C)

摘　　要：目的:调节性T细胞(Treg)是近年来免疫学研究的热点。文中研究获取高纯度Treg的方法及其表型的鉴定。方法:流式细胞术(FACS)分析正常小鼠脾脏中CD4+、CD4+CD25+、CD4+CD25-3个细胞亚群的表型;采用二步磁性细胞分选法(MACS)纯化小鼠脾细胞中CD+CD25+Treg,并对分离后的细胞表型进行鉴定。结果:①依CD25表达水平不同将小鼠天然CD4+T细胞划分为3群,转录因子Foxp3、细胞毒T淋巴细胞相关分子-4(CTLA-4/CD152)、糖皮质激素诱导肿瘤坏死因子受体(GITR)、CD69在CD4+CD25+细胞亚群呈显著性高表达(P<0.01),CD4+CD25+T细胞主要存在于CD45RB低表达细胞亚群,CD28的表达在CD4+CD25+细胞亚群明显下降(P<0.05)。②使用二步MACS法从正常小鼠脾脏中分离CD4+CD25+T淋巴细胞的得率为1%～1.2%,纯度为87.29%～92.04%。FACS分析结果显示,Foxp3优先表达于CD4+CD25+T细胞亚群,表达率为(66.60±1.03)%,而在CD4+和CD4+CD25-T细胞亚群呈低水平表达,分别为(23.97±2.00)%和(16.78±3.24%)。CTLA-4在CD4+CD25+T细胞亚群表达率为(77.37±1.67)%,明显高于在CD4+和CD4+CD25-T细胞亚群的表达[(21.97±2.39)%和(18.04±3.16)%]。纯化的CD4+CD25+T细胞亚群表现为CD127的低表达。结论:MACS阴性加阳性二步分选法避免激惹CD4分子,可获得高纯度且具有天然CD4+CD25+Treg细胞表型及功能特征的细胞。Objective： Regulatory T cells are the hotpoint of immunology for recent years. In this study the preparation of high purity regulatory T cells （Treg） was investisated and the phenotypic and functional characteristics of innate Treg was identified. Methods ： The phenotype of 3-subpopulation of CD4^＋/CD4^＋ CD25^＋/CD4^＋ CD25^-T cells derived from normal murine SPL were analyzed by FACS. CD^＋ CD25^＋ Tregs were isolated by two-step magnetic cell sorting （MACS） and the Treg-specific transcription factor Foxp3, intracellular CTLA-4 and surface antigen CD127 were identified by FACS. Results ： （1）These three CD4^＋ T cell populations defined by varying levels of CD25 expression exhibited marked differences in a number of some intracellular or surface antigens. The CD4^＋ CD25^＋ T cells expressed higher levels of forkhead box P3 （ Foxp3 ） , cytotoxic T lymphocyte associated antigen 4 （ CTLA-4）, glucocortieoid-indueed tumor necrosis factor receptor （ GITR ） and CD69 （P 〈 0.01 ） and lower levels of CD28 （P 〈0.05） than the CD4^＋ or CD4^＋ CD25 subsets. These innate CD4^＋ CD25^＋ Tregs were typically eonfined within the CD45RB^low T cell subset. （2）After CD4^＋ CD25^＋ T eells were isolated from murine splenocytes by 2-step magnetic eell sorting, 90. 19% （range, 87.29% 92.04% ） of the cells expressed both CD4 and CD25, and the yield was 1% - 1.2%. Foxp3 was preferentially expressed by CD4^＋ CD25^＋T cells （66.6±1. 033% ）, whereas CD4^＋ or CD4^＋ CD25- subpopulation eontained low level of Foxp3 （23.97±1. 997% , 16.78±3.24% ）. CTLA4 was expressed by a significantly higher proportion of cells in CD4^＋ CD25^＋ population （77.37±1.67% ） as compared to CD4^＋ （21.97 ±2.39% ） or CD4^＋ CD25^- （ 18.04 ± 3. 16） subsets. The isolated CD4^＋ CD25^＋ T eells also expressed the low intensity of CD127. Conclusion ： These three innate CD4^＋ T-cell populations defined by varying levels of CD25 expression in mice exhibited marke

关 键 词：调节性T细胞 磁性细胞分选法 流式细胞术分析 

分 类 号：R392-33[医药卫生—免疫学]