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首页> 外文期刊>Journal of dermatological science >Development and validation of a new in vitro assay designed to measure contact allergen-triggered oxidative stress in dendritic cells
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Development and validation of a new in vitro assay designed to measure contact allergen-triggered oxidative stress in dendritic cells

机译：一种新的体外测定法的开发和验证，该测定法用于测量树突状细胞中接触变应原触发的氧化应激

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Background: Selected contact allergens are known to induce phenotypic and functional maturation of dendritic cells (DCs). Such changes occurring in DCs have been employed as assay readouts to predict skin-sensitizing potentials of small chemicals. Objective: To respond to the urgent needs for reliable in vitro tests to identify contact allergens, we sought to develop a DC-based assay designed to detect early change(s) induced by sensitizers. Methods: Signature gene expression profiles of skin sensitization were determined by GeneChip and quantitative RT-PCR analyses of RNA samples harvested from mouse skin and XS106 DC line after exposure to dinitrofluorobenzene (DNFB). Production of reactive oxygen species (ROS) was examined indirectly by measuring the level of oxidative stress-XS106 DCs were labeled with a fluorescent dye, CM-H 2DCFDA, exposed to test chemicals, and then examined for fluorescence signals by flow cytometer. Results: DNFB induced abundant mRNA expression of several redox regulatory genes in both mouse skin and XS106DCs. Expression of these genes was inducible by hydrogen peroxide and blocked by a ROS inhibitor, diphenyleneiodonium. Rapid and significant ROS production was induced by 25 of the 28 tested skin sensitizers, but only by 3 of the 21 tested skin irritants. Conclusions: Our small-scale validation study demonstrates the practical utility of our DC-based ROS production assay to detect structurally diverse contact allergens with varying sensitizing potencies. It is tempting to speculate that ROS production in DCs may represent an early event during the sensitization phase.
机译：背景：已知选择的接触过敏原可诱导树突状细胞（DC）的表型和功能成熟。 DC中发生的此类变化已用作测定读数，以预测小型化学品的皮肤致敏潜力。目的：为了响应对可靠的体外测试以识别接触性过敏原的迫切需求，我们寻求开发一种基于DC的测定法，旨在检测敏化剂引起的早期变化。方法：通过GeneChip和定量RT-PCR分析暴露于二硝基氟苯（DNFB）后从小鼠皮肤和XS106 DC系收集的RNA样品，确定皮肤致敏性的特征基因表达谱。通过测量氧化应激水平间接检查了活性氧（ROS）的产生-用荧光染料CM-H 2DCFDA标记XS106 DC，使其暴露于测试化学品，然后通过流式细胞仪检查荧光信号。结果：DNFB在小鼠皮肤和XS106DCs中诱导了多个氧化还原调节基因的mRNA大量表达。这些基因的表达可被过氧化氢诱导，并被ROS抑制剂二苯并碘鎓阻断。测试的28种皮肤敏化剂中有25种引起快速而显着的ROS产生，但测试的21种皮肤刺激剂中只有3种引起。结论：我们的小规模验证研究表明，我们基于DC的ROS生产测定法可检测具有不同敏化力的结构多样的接触变应原，具有实用性。试图推测DC中ROS的产生可能是敏化阶段的早期事件。

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