目的 以3种不同的抗体包被方法，评估人外周血细胞在OKT3刺激前后细胞分泌各种细胞因子的含量，为免疫治疗药物上市前的临床前及临床研究提供理论依据。方法 健康人外周血经密度梯度离心法制备得到外周血单个核细胞（PBMC）后液氮罐中冻存备用，在固相干法、固相湿法（包被24、48 h）及液相法条件下，不同OKT3刺激浓度（1、2 μg·孔^-1）、刺激时间（24、48 h）下，以多功能流式点阵仪（Luminex）检测分泌细胞因子的表达水平。结果 固相干法中OKT3刺激PBMC细胞释放细胞因子显著，OKT3包被48 h、刺激48 h各细胞因子（IL-2、IL-6、IL-10、IFN-γ、TNF-α）分别为阴性对照的6、2、22、214、51倍（1 μg·孔^-1），5、2、26、194、48倍（2 μg·孔^-1）；OKT3包被48 h、刺激24 h各细胞因子（IL-2、IL-6、IL-10、IFN-γ、TNF-α）分别为阴性对照的3、2、2、171、19倍（1 μg·孔^-1），4、2、3、174、21倍（2 μg·孔^-1）；OKT3包被24 h、刺激48 h试验组中，各细胞因子（IL-2、IL-6、IL-10、IFN-γ、TNF-α）分别为阴性对照的6、2、28、356、50倍（1 μg·孔^-1），6、2、35、349、64倍（2 μg·孔^-1）； OKT3包被24 h、刺激24 h各细胞因子（IL-2、IL-6、IL-10、IFN-γ、TNF-α）分别为阴性对照的3、2、1、127、16倍（1 μg·孔^-1），3、1、2、115、13倍（2 μg·孔^-1），表明随着OKT3包被时间延长、与PBMC作用时间延长组别效果更明显；固相湿法与液相法中IL-10呈现明显的含量降低，且二者在释放程度上相当，无组别关系。结论 固相干法孵育系统较固相湿法及液相法有明显的优势，并且OKT3作为阳性对照刺激PBMC诱导人体外细胞因子释放效果显著，可以在药物非临床安全性评价遵循GLP的毒理学试验中使用该方法评估药物引起细胞因子风暴的风险。

Objective Three antibody coating methods were used to evaluate assay of various cytokines secreted by human peripheral blood cells (PBMCs) before and after OKT3 stimulation so as to provide theoretical basis for preclinical and clinical studies of immunotherapy drugs prior to marketing. Methods PBMCs were prepared by healthy human peripheral blood by density gradient centrifugation and cryopreserved in a LN tank for backup. The expression level of cytokines secreted after OKT3 stimulation at different concentrations (1 and 2 μg·well^-1) and time was detected by Luminex based on the solid-phase dry, solid-phase wet (coating for 24 and 48 h), and liquid phase methods. Results In the solid phase immobilized method, OKT3 stimulated PBMC cells to release cytokines significantly. In the group where OKT3 was coated for 48 hours and stimulated for 48 hours, the levels of cytokines (IL-2, IL-6, IL-10, IFN-γ, TNF-α) were 6, 2, 22, 214, and 51 times higher than those of the negative control group (1 μg·well^-1), 5, 2, 26, 194, and 48 times higher (2 μg·well^-1); in the group where OKT3 was coated for 48 hours and stimulated for 24 hours, the levels of cytokines were 3, 2, 2, 171, and 19 times higher than those of the negative control group (1 μg·well^-1), 4, 2, 3, 174, and 21 times higher (2 μg·well^-1); in the group where OKT3 was coated for 24 hours and stimulated for 48 hours, the levels of cytokines were 6, 2, 28, 356, and 50 times higher than those of the negative control group (1 μg·well^-1), 6, 2, 35, 349, and 64 times higher (2 μg·well^-1); in the group where OKT3 was coated for 24 hours and stimulated for 24 hours, the levels of cytokines were 3, 2, 1, 127, and 16 times higher than those of the negative control group (1 μg·well^-1), 3, 1, 2, 115, and 13 times higher (2 μg·well^-1), indicating that the effects were more obvious with longer OKT3 coating time and longer time of interaction between OKT3 and PBMC. In the solid phase wet method and liquid phase method, IL-10 showed a marked decrease in content, and the two methods showed equivalent release levels, with no relationship to the group. Conclusion There was an edge of solid-phase dry method over the other two methods. Significant cytokine release in vitro was observed after OKT3 stimulation as a positive control. Therefore, solid-phase dry method can be used to assess the risk of cytokine storm triggered in GLP toxicology studies.

R965

京津冀基础研究合作专项项目（仿生活性纳米颗粒时序靶向人工血管促进血管再生与相关机制探究）