权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

用于测试环境化学品的毒性和致敏潜力的 3D 皮肤模型

基本信息

批准号：
7936929
负责人：
AKIRA TAKASHIMA
金额：
$ 50万
依托单位：
UNIVERSITY OF TOLEDO HEALTH SCI CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-21 至 2012-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7936929
关键词：
3-Dimensional Address Allergens Allergic Contact Dermatitis Animal Testing Animals Applications Grants Area Atopic Dermatitis Biological Biological Assay Biosensor Cell Line Cell Survival Cells Centers for Disease Control and Prevention (U.S.)Chemicals Collagen Contact Dermatitis Corrosives Cytokine Receptor Gene Dendritic Cells Dermal Detection Development Disease DsRed Economics Engineering Event Exhibits Exposure to Fibroblasts Fluorescence Funding Gel Gene Expression Gene Expression Profiling Gene Proteins Genes Genetic Transcription Growth Human Resources Immune response Incidence Industry Inflammatory Response Intellectual Property International Irritant Dermatitis Irritants Knowledge Langerhans cell Measures Mediating Modeling Occupational Organ Pathogenesis Play Positioning Attribute Proteins Public Health Published Comment Reaction Reagent Regulatory Pathway Reporting Research Research Priority Rodent Role Screening procedure Sensitivity and Specificity Signal Transduction Skin Specificity Stimulus Stress Structure System T-Lymphocyte Technology Testing Toxicity Tests Toxicology United States National Institutes of Health Validation base cost cytokine economic impact environmental chemical health economics high throughput screening improved in vitro Assay in vitro testing in vivo keratinocyte mRNA Expression meetings promoter reconstitution research study response skin irritant transcription factor validation studies virtual

项目摘要

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.

描述（由申请人提供）：本申请解决了广泛的挑战领域（06）使能技术和 06-ES-102 的具体挑战主题：减少研究中动物使用的 3-D 或虚拟模型：创建用于化学毒性测试高通量筛选的微型多细胞器官。尽管毒理学测试已经使用动物进行，但由于公众观点和监管要求的快速变化，迫切需要替代性体外测试。在由环境化学物质引起的众多疾病中，接触性皮炎是发病率最高的疾病——美国疾病预防控制中心将接触性皮炎列为国家职业研究议程（NORA）的21个研究优先领域之一。由于接触性皮炎可以通过消除皮肤与病原体的接触来预防，因此开发用于识别此类病原体的体外测定法对全球公共卫生具有广泛而重大的影响。接触性皮炎分为刺激性皮炎（对刺激性化学物质的先天性炎症反应）和过敏性皮炎（T细胞介导的对皮肤致敏物的适应性免疫反应）。我们已经确定角质形成细胞 (KC) 释放 ATP 是刺激性接触性皮炎发病机制中的初始致病事件。由于已知几种不同的接触性过敏原会诱导皮肤中的树突状细胞 (DC) 表达 IL-1¿ mRNA，因此我们通过对皮肤来源的 DC 系 XS106 进行改造，使其在 IL-1¿ 启动子的控制下表达黄色荧光蛋白 (YFP) 基因，开发了一种 HTP 兼容的 DC 生物传感器克隆。我们的目标是开发一种由 KC、DC 和成纤维细胞 (FB) 组成的 3D 多通道皮肤生物传感器系统，可以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）。在目标 1 下，我们将通过改造 Pam 212 KC 系来构建 KC 生物传感器克隆，以在 Nrf2（一种能够触发 Keep1-Nrf2-ARE 细胞对环境压力的生存反应的转录因子）控制下表达 GFP 基因。我们还将生成在 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）控制下表达 DsRed 基因的第二个 DC 生物传感器克隆。在目标 2 下，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，以及 c) 接种两个 DC 生物传感器克隆以形成具有 KC 的多细胞表皮结构。在目标 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 释放作为皮肤刺激潜力的指标，并测量 YFP、GFP 和 DsRed 信号来识别甚至对皮肤致敏物进行分类。最后，我们将通过在 96 孔板格式中重建 3D 皮肤等效微培养物来提高 HTP 能力。一旦开发并验证，具有双重功能的 3D 皮肤模型可能使该行业能够消除使用动物进行皮肤毒理学研究。在由环境化学物质引起的众多疾病中，接触性皮炎是发病率最高的疾病。我们将开发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可能性。一旦开发并验证，所得的 3D 皮肤模型可能使该行业能够消除使用动物进行皮肤毒理学研究。