人CD4^+CD25^+调节性T细胞体外扩增研究  

In vitro amplification of CD4^+CD25^+ regulatory T cells from the healthy volunteers

作　　者：蓝三春[1] 石蕊[1] 张晓颖[1] 周栋[1] 陈虎[1] 

机构地区：[1]军事医学科学院附属医院造血干细胞移植科,北京100071

出　　处：《中国免疫学杂志》2010年第10期913-918,共6页Chinese Journal of Immunology

基　　金：全军医药卫生科研基金课题(06G124)

摘　　要：目的:建立有效的体外培养扩增人CD4+CD25+Treg细胞的体系,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(Auctegraft versus host disease,aGVHD)提供实验基础。方法:用免疫磁珠分选法从健康人外周血淋巴细胞中得到CD4+T细胞、CD4+CD25-T细胞和CD4+CD25+Treg细胞。分别进行长期培养、扩增;流式分析其表型及FOXP3表达,MTT法检测体外扩增的CD4+CD25+Treg对自体和异体的CD4+T细胞增殖的抑制作用。结果:经过3周培养,三组细胞均有明显的扩增。磁珠分选后的CD4+CD25+Treg细胞的扩增倍数为(20±8)倍,细胞纯度由89.28%±2.43%下降到76.36%±2.38%。CD4+CD25-T细胞组扩增迅速,倍数为(110±21)倍,并且CD4+CD25+Treg细胞的比例由培养前0.55%±0.27%增加到53.73%±3.81%。CD4+T细胞组扩增倍数为(89±8)倍,并且CD4+CD25+Treg细胞的比例由10.63%±3.87%增加到70.93%±1.71%。体外抑制实验显示体外扩增的CD4+CD25+T细胞对自体和异体的CD4+T细胞增殖均有明显的抑制作用,并且随着效靶比浓度的降低而降低。而且其各浓度梯度的抑制作用在自体和异体之间无明显的差异。结论:我们在体外成功扩增了CD4+CD25+Treg细胞,并且具有免疫抑制功能,其中CD4+T细胞组扩增倍数大,细胞数量多,细胞纯度有很大提高,方法简便,有望应用于临床试验。Objective：To establish a practical and convenient approach and experimental foundation for the in vitro expansion of CD4^＋CD25^＋ cells and infusion of donor-derived CD4^＋CD25^＋ cells for future treatment of acute graft versus host disease（aGVHD）. Methods： CD4^＋ T cells, CD ＋ CD25 ^- T cells and CD4 ^＋ CD25 ^＋ T cells were isolated from healthy volunteers by MACS and then expanded in vitro. Phenotype of the T cell lines and expression of the FOXP3 was determined by FACS. The inhibitory effect of expanded CD4^ ＋ CD25^- T cells＇ on auto-and allo CD4^＋ T cells was tested by MTT method. Results： The Treg cells from all the three groups were expanded significantly after three weeks of culture.The proliferation rates in the purity of CD4^＋ CD25^＋ Treg were 20 ± 8 folds. However, the proportion of Treg cells was decreased from 89.28 % ＋ 2.43 % to 76.36% ＋ 2.38 % during the culture. The CD4^＋ CD25^-T cells grew more rapidly and the proliferation rates were increased by 110 ± 21 folds, with the percentage of raising from 0.55 % ± 0.27 % to 53.73 %± 3.81%. In the group of CD4^＋ T cells group, the cells were 89 ＋ 8 folds by with a percentage of Treg cells increasing cells from 10.63 % ± 3.87 % to 70.93 %±1.71%. It was shown by the inhibitory test that the expanded CD4^＋CD25 ^＋T cells could inhibit the proliferation of auto-and allo- CD4 ^＋T cells in vitro in a cell dose-dependent maimer. No significant difference of the inhibition effect was found in auto-and allogroups of Treg cells. Conclusion： We successfully expand CD4^＋ CD25 ^＋ Tregs in vitro. The cells expanded have inhibitory effects in vitro. As the method is simple, we can conveniently obtain lots of high purity of CD4^＋CD25^＋ Tregs from CD4^＋ T cell. Probably in future, the cells may be used in clinical trials.

关 键 词：CD4+CD25+TREG 体外扩增 免疫抑制 

分 类 号：R392.9[医药卫生—免疫学]