目的 以3種不同的抗體包被方法，評(píng)估人外周血細(xì)胞在OKT3刺激前后細(xì)胞分泌各種細(xì)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(gè)核細(xì)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(shí)間（24、48 h）下，以多功能流式點(diǎn)陣儀（Luminex）檢測(cè)分泌細(xì)胞因子的表達(dá)水平。結(jié)果 固相干法中OKT3刺激PBMC細(xì)胞釋放細(xì)胞因子顯著，OKT3包被48 h、刺激48 h各細(xì)胞因子（IL-2、IL-6、IL-10、IFN-γ、TNF-α）分別為陰性對(duì)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(xì)胞因子（IL-2、IL-6、IL-10、IFN-γ、TNF-α）分別為陰性對(duì)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(yàn)組中，各細(xì)胞因子（IL-2、IL-6、IL-10、IFN-γ、TNF-α）分別為陰性對(duì)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(xì)胞因子（IL-2、IL-6、IL-10、IFN-γ、TNF-α）分別為陰性對(duì)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(shí)間延長(zhǎng)、與PBMC作用時(shí)間延長(zhǎng)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(dāng)，無(wú)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(shì)，并且OKT3作為陽(yáng)性對(duì)照刺激PBMC誘導(dǎo)人體外細(xì)胞因子釋放效果顯著，可以在藥物非臨床安全性評(píng)價(jià)遵循GLP的毒理學(xué)試驗(yàn)中使用該方法評(píng)估藥物引起細(xì)胞因子風(fēng)暴的風(fēng)險(xiǎn)。

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(zhuān)項(xiàng)項(xiàng)目（仿生活性納米顆粒時(shí)序靶向人工血管促進(jìn)血管再生與相關(guān)機(jī)制探究）