人外周血CD4^+ CD25^+细胞体外扩增T细胞亚群及功能检测  

Detection of T cell subset and its function of CD4^+ CD25^+ cells expansion in vitro

作　　者：王志红[1] 陈虎[2] 刘广贤[2] 冯凯[2] 廖丽[2] 

机构地区：[1]解放军总医院第一附属医院血液科,北京100037 [2]军事医学科学院附属医院造血干细胞移植科,北京100071

出　　处：《同济大学学报（医学版）》2008年第4期53-57,共5页Journal of Tongji University(Medical Science)

摘　　要：目的建立尼龙面柱法体外分选、扩增CD4+CD25+细胞的方法,并检测扩增后T细胞亚群及其功能,为临床输注供者来源的CD4+CD25+细胞抑制急性移植物抗宿主病(acute graft versus host disease,aGVHD)提供实验基础。方法体外用尼龙棉柱法分离T细胞,加入含Anti-CD35μg/ml、IL-2 500 IU/ml、10%FCS的RPMI-1640培养基进行培养扩增CD4+CD25+细胞,分别在培养前、培养后3、8、16、21、28 d检测T细胞亚群CD3、CD4、CD8,CD4+CD25+及其功能;同时检测CD28+和fasL的表达。结果体外尼龙棉柱法分离的T细胞约16 d后,CD4+CD25+细胞可扩增(50.88±6.21)倍;但CD8+细胞也同时扩增,甚至略多于CD4+CD25+细胞。而T细胞活化指标fasL(介导细胞凋亡的死亡因子)的表达在培养前后的变化无统计学差异。结论尼龙棉柱法在体外扩增培养可获得数量较多的CD4+CD25+Treg细胞,这些细胞不会促进T细胞活化,可能不会增加对宿主细胞的杀伤力。Objective To establish a clinically-usefull, practically-convenient approach of to CD4^＋ CD25^＋ cells expansion in vitro and detect its subset and function, to offer experimental foundation for infusing donorderived CD4^＋ CD25^＋ cells to inhibit acute graft versus host disease （aGVHD）. Methods Peripherial T cells were purified by nylon cotton column,and cultured in RPMI1640 with anti-CD3 （5 μg/ml and IL-2,500 U/ ml）. CD3、 CD4、CD8、CD4^＋ CD25^＋ and its function （CD28^＋ and fasL expression） were detectd by FACS or by RT-PCR. after 3,7,14,21,28 days of culture. Results CD4^＋ CD25^＋ in patients who developed aGVHD is higher compared with that undeveloped. CD4^＋ CD25^＋ cells can expand about 50 folds （50.88 ± 6.21 folds） on 16 day of culture in vitro and adsum statistical significance. CD8^＋ cells raise at the same time. FasL expression change have no statistical significance, and which indicate it cannot raise kill to host.Conclusion After peripherial T cells were purified by nylon cotton column, and cultured,expression of CD4^＋ CD25^＋ ,CD8^＋ will increase,but expression of fasL will not increase,so it is impossibl to raise kill to host.

关 键 词：CD4^+CD25^+细胞 扩增 T细胞亚群 功能 

分 类 号：R552[医药卫生—血液循环系统疾病]