Hofmann Ute, Priem Melanie, Bartzsch Christine, Winckler Thomas, Feller Karl-Heinz
Department of Medical Engineering and Biotechnology, University of Applied Sciences Jena, Carl-Zeiss-Promenade 2, 07745 Jena, Germany.
Department of Pharmaceutical Biology, Institute of Pharmacy, Friedrich-Schiller-University of Jena, Semmelweisstraße 10, 07743 Jena, Germany.
Sensors (Basel). 2014 Jun 25;14(7):11293-307. doi: 10.3390/s140711293.
In the progress of allergic and irritant contact dermatitis, chemicals that cause the generation of reactive oxygen species trigger a heat shock response in keratinocytes. In this study, an optical sensor cell line based on cultured human keratinocytes (HaCaT cells) expressing green fluorescent protein (GFP) under the control of the stress-inducible HSP70B' promoter were constructed. Exposure of HaCaT sensor cells to 25 µM cadmium, a model substance for oxidative stress induction, provoked a 1.7-fold increase in total glutathione and a ~300-fold induction of transcript level of the gene coding for heat shock protein HSP70B'. An extract of Arnica montana flowers resulted in a strong induction of the HSP70B' gene and a pronounced decrease of total glutathione in keratinocytes. The HSP70B' promoter-based sensor cells conveniently detected cadmium-induced stress using GFP fluorescence as read-out with a limit of detection of 6 µM cadmium. In addition the sensor cells responded to exposure of cells to A. montana extract with induction of GFP fluorescence. Thus, the HaCaT sensor cells provide a means for the automated detection of the compromised redox status of keratinocytes as an early indicator of the development of human skin disorders and could be applied for the prediction of skin irritation in more complex in vitro 3D human skin models and in the development of micro-total analysis systems (µTAS) that may be utilized in dermatology, toxicology, pharmacology and drug screenings.
在变应性和刺激性接触性皮炎的进展过程中，导致活性氧生成的化学物质会引发角质形成细胞中的热休克反应。在本研究中，构建了一种基于培养的人角质形成细胞（HaCaT细胞）的光学传感器细胞系，该细胞在应激诱导型HSP70B'启动子的控制下表达绿色荧光蛋白（GFP）。将HaCaT传感器细胞暴露于25µM镉（一种用于诱导氧化应激的模型物质），可使总谷胱甘肽增加1.7倍，并使编码热休克蛋白HSP70B'的基因转录水平诱导增加约300倍。山金车花提取物可强烈诱导角质形成细胞中HSP70B'基因表达，并使总谷胱甘肽显著减少。基于HSP70B'启动子的传感器细胞以GFP荧光作为读出信号，方便地检测镉诱导的应激，检测限为6µM镉。此外，传感器细胞对细胞暴露于山金车提取物的反应是GFP荧光诱导。因此，HaCaT传感器细胞为自动检测角质形成细胞氧化还原状态受损提供了一种手段，作为人类皮肤疾病发展的早期指标，可应用于更复杂的体外3D人体皮肤模型中的皮肤刺激性预测，以及用于皮肤病学、毒理学、药理学和药物筛选的微全分析系统（µTAS）的开发。
