Abstract：
We genetically manipulated HaCaT cells, a spontaneously immortalised normal keratinocyte cell line, to stably express two different coloured luciferase reporter genes, driven by interleukin 8 (IL-8) and ubiquitin-C (UBC) promoters, respectively. Subsequently, we generated a three-dimensional (3D) skin-like in vitro composite (SLIC) utilising these cells, with the objective of monitoring bioluminescence emitted from the SLIC. This SLIC was generated on non-woven silica fibre membranes in differentiation medium. Immunohistochemical analyses of skin differentiation markers in the SLIC revealed the expression of keratins 2 and 10, filaggrin, and involucrin, indicating mature skin characteristics. This engineered SLIC was employed for real-time bioluminescence monitoring, allowing the assessment of time- and dose-dependent responses to UV stress, as well as to hydrophilic and hydrophobic chemical loads. Notably, evaluation of responses to hydrophobic substances has been challenging with conventional 2D cell culture methods, suggesting the need for a new approach, which this technology could address. Our observations suggest that engineered SLIC with constitutively expressing reporters driven by selected promoters which are tailored to specific objectives, significantly facilitates assays exploring the physiological functions of skin cells based on genetic response mechanisms. It also highlights new avenues for evaluating the physiological impacts of various compounds designed for topical application to human skin.
摘要：
我们对HaCaT细胞进行基因操作,自发永生化的正常角质形成细胞系，稳定表达两种不同颜色的荧光素酶报告基因，由白细胞介素8（IL-8）和泛素C（UBC）启动子驱动，分别。随后，我们利用这些细胞生成了三维（3D）皮肤样体外复合材料（SLIC），目的是监测SLIC发出的生物发光。该SLIC在分化培养基中的非织造二氧化硅纤维膜上产生。SLIC中皮肤分化标记的免疫组织化学分析显示角蛋白2和10，聚丝团蛋白的表达，和总蛋白，表明成熟的皮肤特征。该工程SLIC用于实时生物发光监测，允许评估对紫外线胁迫的时间和剂量依赖性反应，以及亲水和疏水化学负载。值得注意的是,用传统的二维细胞培养方法评估对疏水物质的反应一直是一个挑战，表明需要一种新的方法，这项技术可以解决的问题。我们的观察表明，设计的SLIC具有组成型表达报告者，由选定的启动子驱动，这些启动子适合特定的目标，显着促进测定探索基于遗传反应机制的皮肤细胞的生理功能。它还强调了评估为局部应用于人体皮肤而设计的各种化合物的生理影响的新途径。
