Forskolin对小鼠CD8^(+)记忆T细胞富集的影响  

Influence of Forskolin on the enrichment of mouse memory CD8^(+)T cells

作　　者：李铁鹏 王瑶 高耀 何美颉 陈广玉 徐本玲 高全立 LI Tie-peng;WANG Yao;GAO Yao;HE Mei-jie;CHEN Guang-yu;XU Ben-ling;GAO Quan-li(Department of Immunotherapy,Affiliated Cancer Hospital of Zhengzhou University,Zhengzhou,Henan 450008,China)

机构地区：[1]郑州大学附属肿瘤医院免疫治疗科,河南郑州450008

出　　处：《中华实用诊断与治疗杂志》2022年第12期1210-1214,共5页Journal of Chinese Practical Diagnosis and Therapy

基　　金：国家自然科学基金(81902902);河南省医学科技攻关计划省部共建项目(SBGJ202002027);河南省重点研发与推广专项(222102310050,212102310750)。

摘　　要：目的探讨Forskolin对体外培养小鼠不同T细胞亚群的影响及对CD8^(+)记忆T细胞的富集作用。方法25只C57BL/6小鼠,采用密度梯度离心法分离脾脏淋巴细胞,应用CD3^(+)T细胞分离试剂盒分选CD3^(+)T细胞,经体外培养后分为对照组和5、10、20μmol/L Forskolin组,5、10、20μmol/L Forskolin组分别加入5、10、20μmol/L Forskolin,对照组加入等体积DMSO。采用台盼蓝染色法对4组总T细胞数进行计数,应用流式细胞仪检测4组CD8^(+)记忆T细胞比率,选择与对照组差异最大的Forskolin浓度进行后续实验。采用液相色谱-质谱联用法分析对照组和20μmol/L Forskolin组非靶向代谢组学,应用流式细胞仪检测对照组和20μmol/L Forskolin组CD4^(+)T细胞、CD8^(+)T细胞比率及增殖活跃(低CFSE荧光强度)的CD8^(+)记忆、非记忆T细胞比率,计算增殖活跃的CD8^(+)记忆与非记忆T细胞比值(CD8^(+)记忆/非记忆T细胞)。结果对照组和5、10、20μmol/L Forskolin组总T细胞数[(102.91±2.91)%、(69.84±6.88)%、(31.61±2.52)%、(14.02±2.12)%]依次降低(P<0.05);5、10、20μmol/L Forskolin组CD8^(+)记忆T细胞比率[(23.03±12.06)%、(31.58±10.39)%、(44.35±9.10)%]依次升高(P<0.05),对照组[(22.58±6.33)%]与5μmol/L Forskolin组比较差异无统计学意义(P>0.05);选择与对照组差异最大的20μmol/L Forskolin组进行后续实验。对照组与20μmol/L Forskolin组差异代谢物有123种,主成分分析显示2组细胞代谢特征存在差异,20μmol/L Forskolin组三羧酸循环代谢物焦磷酸硫胺素、琥珀酸酯水平均低于对照组(P<0.05),脂肪酸代谢物十六烯酸、鞘磷脂、1-硬脂酰基-2-花生四烯酰基-sn-甘油、二十碳二烯酸水平均高于对照组(P<0.05),糖酵解代谢物α-D-葡萄糖1-磷酸、2-脱氧-D-葡萄糖6-磷酸水平与对照组比较差异均无统计学意义(P>0.05)。20μmol/L Forskolin组CD8^(+)T细胞比率[(91.24±3.71)%]高于对照组[(81.14±9.22)%](t=9.297,P=0.008),CD4^(+)T细胞比率[(0.Objective To investigate the influence of Forskolin on different subsets of mouse T cells during in vitro culture and the enrichment role in memory CD8^(+)T cells.Methods Twenty-five C57BL/6mice were isolated the spleen lymphocytes by density gradient centrifugation.CD3^(+)T cells were sorted using a CD3^(+)T cell isolation kit,and were divided into 5,10and 20μmol/L Forskolin groups as well as control group,which were added with 5,10and 20μmol/L Forskolin,and the same volume of DMSO.Trypan blue staining was used to count T cells in four groups,flow cytometry was used to detect the proportion of memory CD8^(+)T cells,and the Forskolin concentration mostly different from that in control group was selected for subsequent experiments.Non-targeted metabolomics analysis was performed using liquid chromatography-mass spectrometry in control group and 20μmol/L Forskolin group.The proportions of CD4^(+)T cells,CD8^(+)T cells and actively proliferative memory and non-memory CD8^(+)T cells(with low CFSE fluorescence intensity)were detected by flow cytometry in control group and 20μmol/L Forskolin group,and the ratio of memory/non-memory CD8^(+)T cells in active proliferation was calculated.Results The total T cell number decreased gradually in turn in control group and 5,10and 20μmol/L Forskolin groups[(102.91±2.91)%,(69.84±6.88)%,(31.61±2.52)%,(14.02±2.12)%](P<0.05).The proportion of memory CD8^(+)T cells increased gradually in turn in 5,10and 20μmol/L Forskolin groups[(23.03±12.06)%,(31.58±10.39)%,(44.35±9.10)%](P<0.05),and showed no significant difference between control group[(22.58±6.33)%]and 5μmol/L Forskolin group(P>0.05).The concentration of 20μmol/L Forskolin was the mostly different from control group and was selected for the subsequent experiments.There were 123metabolites different between control group and 20μmol/L Forskolin group,and the principal component analysis showed the metabolic characteristics were different between two groups.The levels of the tricarboxylic acid cycle-related metabol

关 键 词：FORSKOLIN 记忆T细胞 增殖 代谢组学 小鼠 

分 类 号：R392[医药卫生—免疫学]