人CD4^+CD25^-CD45RA^+T细胞和CD4^+CD25^-T细胞诱导获得iTreg细胞的方法学研究  

A comparative study of obtaining iTreg by human CD4^+CD25^-CD45RA^+ T cells and CD4^+CD25^- T cells

作　　者：张少鹏 高骥 干晓杰 鞠峥 古鉴 吕凌 ZHANG Shao-Peng;GAO Ji;GAN Xiao-Jie;JU Zheng;GU Jian;L Ling(The First Affiliated Hospital of Nanjing Medical University,Center of Hepatobiliary,Nanjing 210029,China)

机构地区：[1]南京医科大学第一附属医院肝胆中心

出　　处：《中国免疫学杂志》2019年第11期1341-1347,共7页Chinese Journal of Immunology

基　　金：国家高技术研究计划(No.2015AA020932);国家优秀青年基金(No.81522020)资助

摘　　要：目的:分析比较CD4^+CD25 ^-CD45RA^+T细胞和CD4^+CD25^-T细胞在普通/炎性(加入IL-1β和IL-6)条件下,由TGF-β、IL-2和CD3/CD28磁珠共刺激诱导生成iTreg细胞的效率,并对比各组iTreg细胞相关蛋白和细胞因子表达水平变化及其免疫抑制能力的差异。方法:通过Ficoll细胞分离液分离出淋巴细胞,利用MACS分选机,结合CD4阴选磁珠筛出CD4阳性的T细胞,最后再提取其中CD45RA ^+和CD25 ^-T细胞。体外实验中,在普通/炎性条件下诱导iTreg生成,3、6 d通过流式观察各组iTreg细胞的诱导情况及相关分子的表达变化趋势。体内实验分为:CD4 +CD25^-CD45RA ^+T细胞诱导的iTreg组、CD4^+CD25 ^-T细胞诱导的iTreg组和空白对照组,通过观察iTreg对GVHD小鼠模型的免疫调节,分析其免疫抑制能力的差异。结果: CD4^+CD25 -CD45RA +T细胞组和CD4 +CD25 -T细胞组普通条件下iTreg产率分别为(96.00±0.21)%和(60.23±0.50)%,炎性环境下为(81.97±1.29)%和(56.80±0.43)%,前者都显著高于后者( P <0.001)。各组iTreg细胞中CTLA-4、TGF-β和IL-10的表达未见明显差异,而炎性因子IL-2、IL-17前者均低于后者( P <0.001),前者IFN-γ也低于后者( P <0.05)。体内实验表明iTreg均具有免疫调节能力,但CD4^+CD25 ^-CD45RA^+T细胞组诱导的iTreg能力强于后者。结论: CD4^+CD25 ^-CD45RA^+T细胞组和CD4^+CD25^-T细胞组都可以成功诱导iTreg细胞,但是前者在普通和炎性微环境下诱导效率高于后者,免疫调节能力也优于后者。Objective: To analyse the yield rate of iTreg induced from human CD4 +CD25 -CD45RA +T cells and CD4 +CD25 -T cells in normal or inflammatory(add IL-1β/6)conditions under co-stimulation of TGF-β,IL-2 and CD3/CD28 beads,compare the difference on immunosuppressive ability and expression levels of iTreg associated functional proteins or cytokines among groups. Methods: Lymphocytes were obtained through Ficoll lymphocyte separation medium, CD4 +CD25 -CD45RA +T cells and CD4 +CD25 -T cells were isolated by MACS after co-incubation with microbeads.In vitro, yield of iTreg and expression levels of related molecule/cytokines were detected by Flow cytometry on 3 and 6 day respectively.In vivo, GVHD mice were applied to exam the immunosuppressive ability of iTreg, three groups were designed: CD4 +CD25 -CD45RA +T cells derived iTreg group,CD4 +CD25 -T cells derived iTreg group and PBS group. Results: The iTreg yield rate were (96.00±0.21)%,(60.23±0.50)% in normal conditions and (81.97±1.29)%,(56.80±0.43)% in inflammatory conditions,the former was significantly higher than the latter( P <0.001).CTLA-4,TGF-β and IL-10 didn′t show obvious difference while the secretion of pro-inflammatory cytokines such as IL-2,IL-17 and IFN-γ were significantly improved in latter( P <0.05). Conclusion: Study indicated that all iTreg induced in vitro exhibited immunosuppr-essive ability,what′s more,CD4 +CD25 -CD45RA +T cells derived iTregs were stronger in both normal and inflammatory environment.

关 键 词：iTreg CD4 ^+CD25 ^-CD45RA^+T细胞 CD4^+CD25^-T细胞 GVHD 

分 类 号：Q392.3[生物学—遗传学]