保存条件对恒河猴CD8^(+)T细胞活性的影响  

Effects of storage conditions on the activity of CD8^(+)T cells in rhesus monkeys

作　　者：罗嘉慧 卢秋翰 李国萃 张京京 丛喆 魏强 LUO Jiahui;LU Qiuhan;LI Guocui;ZHANG Jingjing;CONG Zhe;WEI Qiang(Comparative Medicine Center,Peking Union College(PUMC)&Institute of Laboratory Animal Science,Chinese Academy of Medical Sciences(CAMS),NHC Key Laboratory of Human Disease Comparative Medicine,Key Laboratory of Human Diseases Animal Models,State Administration of Traditional Chinese Medicine,Beijing Key Laboratory for Animal Models of Emerging and Remerging Infectious Diseases,Beijing 100021,China)

机构地区：[1]北京协和医学院比较医学中心,中国医学科学院医学实验动物研究所,国家卫生健康委员会人类疾病比较医学重点实验室,国家中医药管理局人类疾病动物模型三级实验室,新发再发传染病动物模型研究北京市重点实验室,北京100021

出　　处：《中国比较医学杂志》2021年第4期33-37,共5页Chinese Journal of Comparative Medicine

基　　金：国家自然科学基金(81971944);中国医学科学院医学与健康科技创新工程重大协同创新项目(2017-I2M-1-014);十三五传染病科技重大专项(2017ZX10304402,2018ZX10101001,2018ZX10301103);基础医学学科建设项目——病原生物学。

摘　　要：目的通过细胞内细胞因子染色(intracellular cytokine staining,ICS)检测CD8^(+)T细胞反应强度,探究不同保存条件对CD8+T细胞活性影响。方法采集9只恒河猴外周血,分离外周血单个核细胞(PBMC),一部分新鲜细胞直接用于检测,剩余细胞冻存至-80℃和液氮,冻存1周和1年再用于检测,检测时用阳性刺激物进行刺激,流式分析活细胞比例,MIP-1β、TNF-α、IL-2、IFN-γ四种细胞因子分泌情况和细胞脱粒标志物CD107a的表达情况。结果随着冻存时间的增加,细胞存活率降低,且液氮冻存比-80℃冻存有更高的存活率。在细胞因子分泌和细胞脱粒标志物CD107a的表达情况中,冻存一年的细胞MIP-1β、TNF-α分泌量显著高于正常细胞,细胞状态发生了改变,冻存一周的细胞与新鲜细胞状态更为相近。结论想要保持低的细胞死亡率和正常的CD8+T细胞反应,应使用新鲜细胞或短期冻存的细胞进行测量CD8^(+)T细胞反应水平的实验。Objective To use intracellular cytokine staining to detect the intensity of the CD8+T cell response and explore the effects of different storage conditions on CD8+T cell activity.Methods Rhesus macaque peripheral blood was collected and peripheral blood mononuclear cells were separated.Some fresh cells were used directly for analysis and the remaining cells were frozen at -80℃ or in liquid nitrogen and used for analysis after 1 week or 1 year.A positive stimulus was applied and flow cytometiy was used to analyze the proportion of living cells,secretion of MIP-1β,TNF-α,IL-2,and IFN-γ,and expression of cell degranulation marker CD107a.Results As the storage time was increased,the cell survival rate decreased,and liquid nitrogen freezing had a higher survival rate than freezing at -80℃.Secretion of MIP-1β and TNF-α from cells cryopreserved for 1 year was significantly higher than that from normal cells and the cell status had changed.The status of cells frozen for 1 week was more similar to fresh cells.Conclusions To maintain low cell death and a normal CD8+T cell response,fresh cells or short-term frozen cells should be used for experiments to measure the level of the CD8+T cell response.

关 键 词：细胞内细胞因子染色 CD8^(+)T细胞 细胞活性 恒河猴 

分 类 号：R-33[医药卫生]