体外扩增后脐血来源Treg细胞的有效性及安全性研究  

An Efficacy and Safety Study of Cord Blood-derived,ex vivo-expanded Tregs

作　　者：马小倩[1] 李桑[1] 王维[1] 

机构地区：[1]中南大学湘雅三医院细胞移植与基因治疗中心,中国湖南长沙410013

出　　处：《生命科学研究》2016年第4期333-339,共7页Life Science Research

基　　金：国家自然科学基金(81201171);湖南省卫计委科研计划课题项目(20160125)

摘　　要：通过检测扩增后调节性T细胞(regulatory T cells,Tregs)的有效性和安全性,研究脐血来源的CD4^+CD25^+Foxp3^+Treg细胞的临床运用前景。实验中,新鲜分离的Treg细胞通过CD3/CD28抗体包被微磁珠进行体外扩增,以检测脐血来源Treg细胞的体外增殖能力,同时利用流式细胞术检测新鲜分离的Treg细胞与扩增后细胞的表型。随后,采用混合淋巴实验(mixed lymphocyte reaction,MLR)检测Treg细胞的功能,利用刺激实验检测扩增后Treg细胞的免疫原性,并且利用NOD-Prkdc^(scid)IL2rg^(null)免疫缺陷小鼠进行体内安全性检测。结果显示,4周后脐血来源的Treg细胞能够扩增到1 000倍以上,与新鲜分离的Treg细胞相比,扩增后的细胞CD45RA表达降低,CD45RO和CD39表达升高;同时,MLR结果显示扩增后的Treg细胞具有较强的抑制能力,并且免疫原性低;此外,体内结果显示扩增后的Treg细胞不会引起移植物抗宿主反应。实验结果初步表明,脐血来源体外扩增后的Treg细胞不仅在数量和功能上能够满足临床移植的需要,并且免疫原性低安全性高。To investigate the potential clinical application of cord blood （CB）-derived regulatory T cells （Tregs）, the efficacy and safety of third-party, ex vivo-expanded, CB-derived Tregs were detected. In experiment, freshly isolated Tregs were cultured with anti-CD3/CD28 microbeads in vitro for 4 weeks. Phenotypic char- acteristics of the freshly isolated Tregs and the expanded Tregs were examined by fluorescence activated cell sorting （FACS）. The mixed lymphocyte reaction （MLR） was used to identify the function of Tregs in vitro, and the stimulation assay was used to determine the immunogenicity of Tregs from CB. Also NOD-Prkdcid IL2rgruli mice were used to test the safety in vivo. The results showed that, after expansion for 4 weeks, the number of CB-derived Tregs increased more than 1 000 fold. The FACS showed that the expanded Tregs still retained CD4＋CD25＋Foxp3＋CD127low phenotype, but had significantly increased expressions of CD45RO and CD39 when compared with the freshly isolated Tregs. MLR assay also demonstrated that the expanded CB-derived Tregs had significantly higher suppression ability compared to the freshly isolated Tregs. Stimu- lation assay showed the expanded Tregs still had low immunogenicity and in vivo studies confirmed that the expanded Tregs did not induce GVHD. The primary data demonstrated that, after expansion, CB-derived Tregs could meet the needs of clinical transplantation not only for enough number and good suppression a- bility but also for their low immunogenicity and good safety.

关 键 词：调节性T细胞(Tregs) 脐血(CB) 体外扩增 抑制功能 有效性 安全性 

分 类 号：R392.4[医药卫生—免疫学]