摘要
目的研究羧胺三唑(CAI)对CD8+T细胞的作用,并探索其增强活化的T细胞杀伤作用的关键机制。方法用免疫磁珠分选出小鼠CD8+T细胞,分为对照组、CAI组、ZK756326(Ca2+激活剂)组以及CAI+ZK756326组。流式细胞计量术检测细胞内游离钙离子水平;酶联免疫吸附法检测细胞内钙调磷酸酶(CaN)的表达;免疫荧光染色检测细胞活化T细胞核因子2(NFAT2)核转运;染色质免疫共沉淀-qPCR检测细胞中NFAT2调控程序性死亡受体1(PD-1)表达。分离小鼠脾脏细胞毒性T淋巴细胞(CTLs),流式细胞计量术检测其中CD8+T细胞PD-1的表达。结果CAI组显著降低CD8+T细胞内Ca2+浓度(P<0.001),CAI+ZK756326组胞内Ca2+浓度有所升高(P<0.01);CAI显著降低CD8+T细胞中钙调磷酸酶的含量(P<0.001);CAI可显著抑制NFAT2核转运(P<0.001),并使NFAT2依赖的PD-1转录进程显著降低(P<0.001);CAI使小鼠脾脏CTLs细胞中PD-1+CD8+T细胞比例显著减少(P<0.001)。结论CAI通过抑制CD8+T细胞内钙离子水平以及钙调磷酸酶的表达抑制NFAT2核转运,从而降低CD8+T细胞PD-1的表达,进一步产生免疫治疗干预作用。
Objective To study the effect of carboxyamidotriazole(CAI)on CD8+T cells,and to explore the critical mechanism of CAI in reinforcing cytotoxicity of activated T cells.Methods CD8+T cells of mouse were isolated by immunomagnetic beads and divided into control group,CAI group,ZK756326(Ca2+activator)group and CAI+ZK756326 group.Flow cytometry was used to detect the level of intracellular free calcium ions in CD8+T cells;Enzyme-linked immunosorbent assay was used to detect the expression of intracellular calcineurin(CaN);Immunoflu-orescence staining was used to detect NFAT2 nuclear transportation;Chromatin immunoprecipitation-qPCR was used to detect the expression of PD-1 regulated by NFAT2.Mice spleen cytotoxic T lymphocytes(CTLs)were isolated and examined for the expression of PD-1 in CD8+T cells by flow cytometry.Results Intracellular Ca2+concentration in CD8+T cells significantly decreased in the CAI group(P<0.001),while intracellular Ca2+concentration increased in the CAI+ZK756326 group(P<0.01);CAI significantly decreased the content of calcineurin phosphatase in CD8+T cells(P<0.001);CAI significantly inhibited NFAT2 nuclear transportation(P<0.001)and significantly inhibited PD-1 transcription process depend on NFAT2(P<0.001);CAI significantly reduced the proportion of PD-1+CD8+T cells in mice spleen CTLs cells(P<0.001).Conclusions CAI inhibits NFAT2 nuclear translocation by decreasing the level of calcium ions in the CD8+T cells and inhibiting the expression of calcineurin,thereby reducing the expression of PD-1 in CD8+T cells,which facilitated immunotherapy intervention.
作者
许梦姣
高洪婷
石婧
鞠瑞
杨黎星
李建恒
郭磊
XU Meng-jiao;GAO Hong-ting;SHI Jing;JU Rui;YANG Li-xing;LI Jian-heng;GUO Lei(Department of Pharmacy,Hebei University,Baoding 071002;Department of Pharmacology,Institute of Basic Medical Sciences CAMS, School of Basic Medicine PUMC,Beijing 10005,China)
出处
《基础医学与临床》
CSCD
2020年第5期627-631,共5页
Basic and Clinical Medicine
基金
国家自然科学基金(81872897)
中国医学科学院医学与健康科技创新工程(2016-I2M-1-011)
北京协和医学院研究生创新基金(2017-1007-01)。
