结核性胸液中NK细胞的活化、记忆表型及分泌IFN-γ功能的多色流式分析  

Activation, memory associated molecules analysis and IFN-γ secretion of NK cells from TB pleural fluids analyzed by multi-color flow cytometry

作　　者：付笑迎[1] 陈运生[1] 吴长有 FU Xiaoying;CHEN Yunsheng;WU Changyou(Department of Clinical Laboratory,Shenzhen Children’s Hospital,Shenzhen 518038,China;Institution of Immunology,Zhongshan School of Medicine,Sun Yat-sen University,Guangzhou 510080,China)

机构地区：[1]深圳市儿童医院检验科,518038 [2]中山大学医学院免疫学研究所,广州510080

出　　处：《免疫学杂志》2020年第8期690-695,共6页Immunological Journal

基　　金：国家自然科学基金(81801588);广东省医学科学技术研究基金(A2018329);广东省组织器官区域免疫与重点实验室开放基金(2019B030301009-006)。

摘　　要：目的探讨结核性胸液细胞(pleural fluid cells,PFCs)中NK细胞的活化标志、记忆相关表型、NK细胞表面受体表达以及分泌细胞因子IFN-γ的情况。方法以正常人外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)作对照,应用多色流式细胞术分析结核性胸液中NK细胞活化及记忆相关表面分子的表达情况,同时分析在细胞因子IL-12的作用下结核性胸液中NK细胞分泌IFN-γ的情况。结果与PBMCs中的NK细胞相比,PFCs中的NK细胞表达高水平早期活化标志CD69,并表达T细胞记忆相关分子CD45RO。进一步分析NK细胞的记忆相关表型,约50%的CD45RO^+NK细胞同时表达CD27,而几乎99%的CD45RO^+NK细胞不表达CD62L。PFCs中约7.3%的NK细胞同时表达CD45RO和NKG2D,约2.4%的NK细胞同时表达CD45RO和NKG2A。NK细胞功能实验提示在细胞因子IL-12的作用下,CD45RO^+NK细胞分泌更高水平的IFN-γ。结论结核性胸液中NK细胞具有记忆细胞的表型特征,表现出CD45RO^+CD27^+CD62L-效应型记忆细胞特征,在IL-12因子刺激下,CD45RO^+NK细胞产生更高水平的IFN-γ,可能在预防结核感染中发挥重要作用。This study was designed to characterize the activation and memory associated molecules expression on NK cells from human tuberculosis(TB)pleural fluids,and to analyze the production of IFN-γby NK cells in response to IL-12.Pleural fluid cells(PFCs)were isolated from pleural fluids,while PBMCs isolated from normal individuals were used as control.Surface markers of PFCs and PBMCs were stained with monoclonal antibodies and analyzed at the single cell level by multi-color flow cytometry.IFN-γsecretion by NK cells from PFCs was analyzed under the stimulation of IL-12.Compared with NK cells from PBMCs,higher expression of CD69 and memory associated molecule CD45 RO was observed on NK cells from PFCs,and about 10.7%NK cells from PFCs coexpressed CD69 and CD45 RO.Further analysis of other memory associated markers showed that there were high expression of CD27 and CD62 L on NK cells from PFCs,and about 50%CD45 RO^+NK cells co-expressed CD27,while almost CD45 RO^+NK cells expressed no CD62 L.Meanwhile,around 7.3%NK cells from PFCs co-expressed CD45 RO and NKG2 D,and 2.4%NK cells from PFCs co-expressed CD45 RO and NKG2 A.CD45 RO^+NK cells expressed higher percentage of IFN-γcompared with CD45 RO-NK cells in response to IL-12.In conclusion,NK cells from PFCs are highly expressed CD69 and CD45 RO,which are associated memory T cells.The result also indicated that NK cells from PFCs may have a memory phenotype.Phenotypic analysis indicated that CD45 RO^+NK cells are CD45 RO^+CD62 L-effector memory phenotype,and produce higher level of IFN-γin response to IL-12,suggesting these cells probably play essential role in preventing TB infection.

关 键 词：NK细胞 结核性胸膜炎 免疫记忆 

分 类 号：R392.12[医药卫生—免疫学]