3D Skin Model to Test Toxic and Sensitizing Potentials of Environmental Chemicals

描述（由申请人提供）：本申请涉及广泛的挑战领域（06）使能技术和06- es -102的特定挑战主题：减少研究中动物使用的3d或虚拟模型：用于高通量筛选化学毒性测试的微型多细胞器官的创建。虽然已经使用动物进行了毒理学试验，但由于公众观点和监管要求的迅速变化，迫切需要进行替代性体外试验。在环境化学物质引起的多种疾病中，接触性皮炎是发病率最高的疾病——美国疾病控制与预防中心已将接触性皮炎列为美国国家职业研究议程（NORA）的21个研究重点领域之一。由于可以通过消除皮肤与病原体的接触来预防接触性皮炎，因此开发用于鉴定这些病原体的体外测定法对全球公共卫生具有广泛和重大的影响。接触性皮炎分为刺激性皮炎（对刺激性化学物质的先天炎症反应）和过敏性皮炎（T细胞介导的对皮肤增敏剂的适应性免疫反应）。我们已经确定ATP释放角化细胞（KCs）在刺激性接触性皮炎发病机制的初始和致病事件。由于已知几种不同的接触过敏原可诱导皮肤树突状细胞（DC）表达IL-1¿mRNA，我们通过对皮肤来源的DC系XS106进行工程设计，在IL-1¿启动子的控制下表达黄色荧光蛋白（YFP）基因，开发了一个与htp兼容的DC生物传感器克隆。我们的目标是开发一种3D多通道皮肤生物传感器系统，该系统由KCs， dc和成纤维细胞（FBs）组成，并允许同时检测皮肤刺激物（通过ATP释放测量）和皮肤致敏物（通过荧光信号测量）。在Aim 1下，我们将通过对Pam 212 KC细胞系进行工程改造，使其在Nrf2（一种能够触发Keep1-Nrf2-ARE细胞对环境胁迫的存活反应的转录因子）的控制下表达GFP基因，构建KC生物传感器克隆。我们还将在Tfec（一种已知调节细胞因子和细胞因子受体基因表达的转录因子）的控制下生成第二个表达DsRed基因的DC生物传感器克隆。在目标2中，我们将通过以下方式重建3D皮肤模型：a)在水合胶原凝胶中培养NS FB系以形成真皮等效基质，b)在胶原基质顶部培养KC生物传感器克隆，c)播种两个DC生物传感器克隆以形成具有KCs的多细胞表皮结构。在Aim 3下，我们将在生成的3D皮肤模型中测试18种皮肤刺激物和36种接触致敏剂，以评估敏感性和特异性。我们将测量ATP释放作为皮肤刺激电位和YFP， GFP和DsRed信号的指标，以识别甚至分类皮肤致敏剂。最后，我们将通过在96孔板格式中重建3D皮肤等效微培养来提高HTP能力。一旦开发和验证，具有双重功能的3D皮肤模型可能使行业消除使用动物进行皮肤毒理学研究。在环境化学物质引起的多种疾病中，接触性皮炎是发病率最高的疾病。我们将开发一个三维、多细胞皮肤培养系统，以测试大量工业和环境化学品引起刺激性和过敏性接触性皮炎的潜力。一旦开发和验证，由此产生的3D皮肤模型可能使该行业不再使用动物进行皮肤毒理学研究。

DESCRIPTION (provided by applicant): This application addresses the broad challenge area (06) Enabling Technologies and the specific Challenge Topic of 06-ES-102: 3-D or Virtual Models to Reduce Use of Animals in Research: Creation of Miniature Multi- Cellular Organs for High Throughput Screening for Chemical Toxicity Testing. Although toxicology tests have been performed using animals, there exists an urgent and critical need for alternative in vitro tests due to rapid changes in public views and regulatory requirements. Among many diseases caused by environmental chemicals, contact dermatitis is the leading disease in terms of the incidence - the CDC has listed contact dermatitis as one of the 21 research priority areas of the National Occupational Research Agenda (NORA). Because contact dermatitis is preventable by eliminating skin contact with causative agents, development of in vitro assays for identification of such agents has a broad and major impact on the global public health. Contact dermatitis is classified into irritant dermatitis (innate inflammatory responses to irritant chemicals) and allergic dermatitis (T cell-mediated adaptive immune responses to skin sensitizers). We have identified ATP release from keratinocytes (KCs) as an initial and causative event in the pathogenesis of irritant contact dermatitis. Because several different contact allergens are known to induce IL-1¿ mRNA expression by dendritic cells (DCs) in the skin, we have developed a HTP-compatible DC biosensor clone by engineering our skin-derived DC line XS106 to express the yellow fluorescence protein (YFP) gene under the control of the IL-1¿ promoter. Our objective is to develop a 3D multi-channel skin biosensor system that is composed of KCs, DCs, and fibroblasts (FBs) and that allows simultaneous detection of skin irritants (measured by ATP release) and skin sensitizers (measured by fluorescence signals). Under Aim 1, we will construct a KC biosensor clone by engineering the Pam 212 KC line to express the GFP gene under the control of Nrf2 (a transcription factor capable of triggering the Keep1-Nrf2-ARE cell survival response to environmental stresses). We will also generate a second DC biosensor clone expressing the DsRed gene under the control of Tfec (a transcription factor known to regulate cytokine and cytokine receptor gene expression). Under Aim 2, we will reconstitute a 3D skin model by: a) culturing the NS FB line in hydrated collagen gel to form a dermal-equivalent matrix, b) culturing a KC biosensor clone on the top of the collagen matrix, and c) seeding two DC biosensor clones to form a multi-cellular epidermal structure with KCs. Under Aim 3, we will test 18 skin irritants and 36 contact sensitizers in the resulting 3D skin model to evaluate the sensitivity and specificity. We will measure ATP release as an indicator of skin irritating potentials and YFP, GFP, and DsRed signals to identify and even classify skin sensitizers. Finally, we will improve the HTP capability by reconstituting 3D skin-equivalent micro- cultures in a 96 well-plate format. Once developed and validated, the 3D skin model with dual functionality may enable the industry to eliminate the use of animals for skin toxicology studies. Among many diseases caused by environmental chemicals, contact dermatitis is the leading disease in terms of the incidence. We will develop a three-dimensional, multi-cellular skin culture system to test a large number of industrial and environmental chemicals for their potentials to cause irritant and allergic contact dermatitis. Once developed and validated, the resulting 3D skin model may enable the industry to eliminate the use of animals for skin toxicology studies.