细胞因子释放综合征的体外试验探索性研究  

Exploratory study of cytokine release syndrome in vitro

作　　者：傅盈双 李双星 姜华[1] 屈哲[1] 霍桂桃[1] 杨艳伟[1] 张頔[1] 黄瑛[1] 李波[1] 林志[1] FU Yingshuang;LI Shuangxing;JIANG Hua;QU Zhe;HUO Guitao;YANG Yanwei;ZHANG Di;HUANG Ying;LI Bo;LIN Zhi(National Center for Safety Evaluation of Drugs,Beijing Key Laboratory for Nonclinical Safety Evaluation of Drugs,Institutefor Safety Evaluation and Research,NIFDC,Beijing 100176,China)

机构地区：[1]国家药物安全评价监测中心,药物非临床安全性评价研究北京市重点实验室,中国食品药品检定研究院安全评价研究所,北京100176

出　　处：《中国药物警戒》2024年第6期625-631,共7页Chinese Journal of Pharmacovigilance

基　　金：国家重点研发计划(2021YFA1101602);中国食品药品检定研究院学科带头人课题项目(2023X3)。

摘　　要：目的建立细胞因子释放综合征(CRS)的体外评价方法,对试验体系细胞密度、阳性药种类及浓度进行探索。方法分别用CD3抗体(OKT3)(0.1~5μg)、OKT3(0.1~1μg)+CD28抗体(anti-CD28)(1、2μg·mL^(-1))、佛波酯(PMA)(10~100 ng·mL^(-1))、PMA(10~100 ng·mL^(-1))+离子霉素(ION)(1、5μg·mL^(-1))刺激外周血单个核细胞(PBMC)48 h,用CCK-8法检测细胞增殖情况来筛选PBMC的最佳密度及药物的最佳浓度。实验分为7组:对照组、OKT30.1μg+anti-CD281μg·mL^(-1)组、OKT30.5μg+anti-CD281μg·mL^(-1)组、OKT31μg+anti-CD281μg·mL^(-1)组、PMA 10 ng·mL^(-1)+ION 1μg·mL^(-1)组、PMA 25 ng·mL^(-1)+ION 1μg·mL^(-1)组、PMA 50 ng·mL^(-1)+ION 1μg·mL^(-1)组,分别与PBMC细胞暴露48 h,收集细胞上清,通过酶联免疫吸附试验(ELISA)检测药物作用下PBMC释放细胞因子白介素-6(IL-6)、干扰素γ(IFN-γ)情况。结果与对照组相比,OKT3和PMA均引起中密度、高密度PBMC显著增殖,各剂量OKT3和PMA均引起细胞增殖。与阳性药单独使用相比,药物联合使用产生更强的细胞活化效应。与对照组相比,在OKT3(0.1~1μg)+anti-CD28(1μg·mL^(-1))作用下,PBMC释放IL-6、IFN-γ显著增加;在PMA(10~50 ng·mL^(-1))+ION(1μg·mL^(-1))作用下,PBMC分泌IFN-γ与对照组相比显著增加,在PMA(25~50 ng·mL^(-1))+ION(1μg·mL^(-1))作用下,PBMC分泌IL-6与对照组相比显著增加。结论确定了PBMC体外CRS风险评价方法的细胞密度、阳性药物种类及浓度、药物联合使用浓度。Objective To establish an in vitro evaluation method of cytokine release syndrome(CRS),and explore the cell density,positive drug types and concentrations of the test system.Methods Peripheral blood mononuclear cells(PBMC)were stimulated with CD3 antibody(OKT3)(0.1~5μg),OKT3(0.1~1μg)+CD28 antibody(anti-CD28)(1 and 2μg·mL^(-1)),phorbol-12-myristate-13-acetate(PMA)(10~100 ng·mL^(-1)),and PMA(10~100 ng·mL^(-1))+ionomycin(ION)(1 and 5μg·mL^(-1))for 48 h,respectively.The proliferation of PBMC was assessed by the CCK-8 method to determine the optimal cell density and drug concentration.There were seven groups in the experiment:Control group,OKT30.1μg+anti�CD281μg·mL^(-1)group,OKT30.5μg+anti-CD281μg·mL^(-1)group,OKT31μg+anti-CD281μg·mL^(-1)group,PMA 10 ng·mL^(-1)+ION 1μg·mL^(-1)group,PMA 25 ng·mL^(-1)+ION 1μg·mL^(-1)group and PMA 50 ng·mL^(-1)+ION 1μg·mL^(-1)group.The cell supernatant was collected after 48 h of exposure to the respective treatments and cytokine IL-6 and IFN-γwas detected by ELISA.Results Compared with the control group,both OKT3 and PMA induced significant proliferation of medium density and high density PBMC,and OKT3 and PMA induced cell proliferation at all doses.The combination of drugs produced a stronger cell activation effect than the positive drug alone.Compared with the control group,the release of IL-6 and IFN-γfrom PBMC was significantly increased under OKT3(0.1~1μg)+anti-CD28(1μg·mL^(-1))treatment.Under PMA(10~50 ng·mL^(-1))+ION(1μg·mL^(-1)),the secretion of IFN-γby PBMC was significantly increased compared with the control group,and under PMA(25-50 ng·mL^(-1))+ION(1μg·mL^(-1)),the secretion of IL-6 by PBMC was significantly increased compared with the control group.Conclusion The study determines the cell density,positive drug types and concentrations,and drug combination of CRS risk assessment methods for PBMC in vitro.

关 键 词：细胞因子释放综合征 CD3单克隆抗体 CD28抗体 佛波酯 离子霉素 外周血单个核细胞 细胞密度 

分 类 号：R965.3[医药卫生—药理学]