CD137分子在 HIV 特异性 T 淋巴细胞单克隆分选和扩增中的应用  

CD137 molecule permits single cell sorting and expansion of HIV-specific T cells in vitro

作　　者：李丹[1,2] 梁华[1] 鞠斌[1] 范瑾[1] 李亚锋[1] 王硕[1] 邵一鸣 

机构地区：[1]中国疾病预防控制中心性病艾滋病预防控制中心传染病预防控制国家重点实验室,北京102206 [2]感染性疾病诊治协同创新中心,杭州310003

出　　处：《中华微生物学和免疫学杂志》2015年第4期305-309,共5页Chinese Journal of Microbiology and Immunology

摘　　要：目的：建立通过CD137分子对HIV特异性T淋巴细胞进行单克隆分选和体外扩增的方案，对扩增后T细胞的数量和表型进行鉴定。方法分离HIV感染者外周血单个核细胞，采用多色流式细胞染色技术，分离HIV Gag特异性CD3＋CD8＋CD137＋T细胞，单个细胞分选入96孔板，在体外加入饲养细胞和细胞因子进行培养，克隆扩增后14 d对扩增的细胞进行表型鉴定，14 d和28 d分别计算扩增倍数。结果 CD137分子在未接受抗原刺激的状态下表达量很低， Gag肽库刺激后， CD137表达升高，并且表达CD137的CD8细胞均分泌IFN-γ。体外扩增14 d可以获得106的CD8 T细胞，28 d可获得107～108的 CD8 T细胞。在 Gag 肽库刺激后可达到接近100％的活化。结论CD137可替代IFN-γ等传统的细胞因子对HIV抗原特异性T细胞进行筛选和体外扩增，扩增后的T细胞仍保持非常好的活性状态。Objective To isolate HIV-specific T cell clone and to expand them in vitro through the activation-induced expression of CD137 molecule.Methods The peripheral blood mononuclear cells were isolated from HIV-infected patients and HIV Gag specific CD3＋CD8＋CD137＋T cell subset were sorted to 96-well plate in 1 cell/well by multicolor flowcytometry and single cell sorting.After 14 days in vitro culture with feeder cells and cytokines, the numbers and phenotypes of the cultured HIV-specific T cells were calcu-lated and identified.Results The CD137 expression was low on rested T cells but up regulated by the stim-ulation with Gag peptide pool.The CD8＋CD137＋T cells could secret IFN-γ.The number of CD8 T cells reached to 106 after 14 days in culture and expanded to 107-108 cells after 28 days of culture in vitro 100%of the cells remained activated upon Gag stimulation.Conclusion In stead of using IFN-γ, CD137 could be utilized as a novel molecule to isolate and expand HIV specific T cells in vitro.The expanded antigen spe-cific T cell clones could maintain good activation status.

关 键 词：CD137 HIV 单克隆分选 体外扩增 

分 类 号：R392[医药卫生—免疫学]