目的 以3種不同的抗體包被方法，評估人外周血細胞在OKT3刺激前后細胞分泌各種細胞因子的含量，為免疫治療藥物上市前的臨床前及臨床研究提供理論依據(jù)。方法 健康人外周血經(jīng)密度梯度離心法制備得到外周血單個核細胞（PBMC）后液氮罐中凍存?zhèn)溆?，在固相干法、固相濕法（包?4、48 h）及液相法條件下，不同OKT3刺激濃度（1、2 μg·孔^-1）、刺激時間（24、48 h）下，以多功能流式點陣儀（Luminex）檢測分泌細胞因子的表達水平。結(jié)果 固相干法中OKT3刺激PBMC細胞釋放細胞因子顯著，OKT3包被48 h、刺激48 h各細胞因子（IL-2、IL-6、IL-10、IFN-γ、TNF-α）分別為陰性對照的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各細胞因子（IL-2、IL-6、IL-10、IFN-γ、TNF-α）分別為陰性對照的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試驗組中，各細胞因子（IL-2、IL-6、IL-10、IFN-γ、TNF-α）分別為陰性對照的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各細胞因子（IL-2、IL-6、IL-10、IFN-γ、TNF-α）分別為陰性對照的3、2、1、127、16倍（1 μg·孔^-1），3、1、2、115、13倍（2 μg·孔^-1），表明隨著OKT3包被時間延長、與PBMC作用時間延長組別效果更明顯；固相濕法與液相法中IL-10呈現(xiàn)明顯的含量降低，且二者在釋放程度上相當，無組別關(guān)系。結(jié)論 固相干法孵育系統(tǒng)較固相濕法及液相法有明顯的優(yōu)勢，并且OKT3作為陽性對照刺激PBMC誘導人體外細胞因子釋放效果顯著，可以在藥物非臨床安全性評價遵循GLP的毒理學試驗中使用該方法評估藥物引起細胞因子風暴的風險。

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

京津冀基礎(chǔ)研究合作專項項目（仿生活性納米顆粒時序靶向人工血管促進血管再生與相關(guān)機制探究）