利用THP-1细胞进行致敏物鉴定的方法验证

发布时间：2018-07-17 17:55

【摘要】：目的探讨建立以THP-1源性巨噬细胞为受试细胞,以细胞上清液中细胞因子表达改变为检测指标的体外检测化学物致敏性的试验方法。方法将人单核细胞株THP-1细胞用佛波酯(PMA,0.1μg/ml)诱导,使之转化为具有贴壁能力的THP-1巨噬细胞。选择5种已知的化学致敏物硫酸镍(NiSO4)、2,4-二硝基氯代苯(DNCB)、苯二胺(PPDA)、重铬酸钾(K2Cr2O7)、异氰酸酯(TDI)和2种非化学致敏物十二烷基磺酸钠(SDS)、异丙醇(IPA)为受试物,检测不同浓度受试物处理细胞的不同时间点时细胞因子表达的变化。分别用ELISA法检测细胞培养上清IL-6、TNF-α的水平,用流式液相多重蛋白定量技术(CBA)检测IL-1β、IL-8细胞因子的表达水平。结果 NiSO4、DNCB和K2Cr2O7作用6h,PPDA、TDI作用12h时,THP-1细胞上清中IL-6、TNF-α、IL-1β和IL-8四种细胞因子的表达量最高。5种化学致敏物NiSO4、DNCB、K2Cr2O7、PPDA和TDI在最适时间点最适浓度刺激细胞分泌IL-6的水平分别为对照组的40、25、20、50和50倍,TNF-α水平为对照组的20、12、20、8和5倍,IL-1β水平为对照组的30、60、25、30和45倍,IL-8水平为对照组的15、12、15、12和7倍;非致敏物SDS与IPA处理组IL-6水平均有升高,其中SDS组升高最明显,约为空白对照组的20倍。TNF-α、IL-1β和IL-8表达水平无明显变化。结论利用THP-1巨噬细胞检测细胞因子TNF-α、IL-1β和IL-8表达水平的试验方法可以有效区分化学致敏物与非致敏物,因此这3个指标可作为鉴别化学致敏物和非致敏物的有效参考指标。
[Abstract]:Objective to establish an in vitro method for the detection of chemosensitivity of THP-1 derived macrophages and the change of cytokine expression in the supernatant. Methods THP-1 cells were induced by PMA-0.1 渭 g/ml and transformed into adherent THP-1 macrophages. Five known chemical sensitizers nickel sulfate (NiSO4) 2-dinitrochlorobenzene (DNCB), phenylenediamine (PPDA), potassium dichromate (K _ 2Cr _ 2O _ 7), isocyanate (TDI), sodium dodecyl sulfonate (SDS) and isopropanol (IPA) were selected. The cytokine expression was measured at different time points in different concentrations of the cells treated with different concentrations. The level of IL-6 TNF- 伪 in the supernatant of cell culture was detected by Elisa, and the expression of IL-8 cytokines was detected by flow liquid phase multiplex protein quantitative assay (CBA). Results the expression of IL-6, TNF- 伪, IL-1 尾 and IL-8 in supernatant of THP-1 cells was the highest in the supernatant of THP-1 cells treated with DNCB and K2Cr2O7 for 6 h after TDI treatment for 12 h. The levels of IL-6 secreted by cells stimulated by NiSO4 and DNCBC2O7PPDA and TDI at the optimal concentration were the highest in the control group, respectively. The levels of TNF- 伪 in the control group were 50 and 50 times higher than those in the control group. The levels of IL-1 尾 in the control group were 30 and 45 times higher than those in the control group. The levels of IL-8 in the control group were 15, 12, 15, 12 and 7 times higher than those in the control group, and the levels of IL-1 尾 in the control group were 30 and 45 times higher than those in the control group. The level of IL-6 increased in both SDS and IPA groups, and the level of IL-1 尾 and IL-8 in SDS group was significantly higher than that in the control group, and the expression of IL-1 尾 and IL-8 in SDS group was not significantly changed as compared with that in the blank control group. Conclusion the method of detecting the expression level of cytokine TNF- 伪, IL-1 尾 and IL-8 by THP-1 macrophages can effectively distinguish chemosensitizer from non-sensitizer, so these three indexes can be used as effective reference indexes for distinguishing chemosensitizer from non-sensitizer.
【作者单位】：河北联合大学公共卫生学院;中国疾病预防控制中心职业卫生与中毒控制所化学污染与健康安全重点实验室;
【基金】：“十二五”国家科技支撑计划课题(No.2014BAl12B02) 国家自然科学基金重点项目(No.81130050)
【分类号】：R758.22

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