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（由应用程序提供）：此应用程序地址为启用技术的广泛挑战领域（06）和06-ES-102：3-D或虚拟模型的特定挑战主题，以减少动物在研究中的使用：创建微型多细胞器官以进行化学毒性测试的高吞吐量筛选。尽管已经使用动物进行了毒理学测试，但由于公众观点和法规要求的迅速变化，对替代体外测试的急需和迫切需要。在环境化学物质引起的许多疾病中，接触性皮炎是事件的主要疾病 - 疾病预防控制中心已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。由于可以通过消除与严重药物的皮肤接触来预防接触性皮炎，因此开发用于鉴定这种药物的体外测定法对全球公共卫生产生了广泛而重大的影响。接触性皮炎分为刺激性皮炎（对刺激性化学物质的先天炎症反应）和过敏性皮炎（T细胞介导的适应性免疫血液对皮肤传感器）。我们已经将ATP从角质形成细胞（KCS）释放为刺激性接触性皮炎发病机理的初始事件。由于已知多种不同的接触过敏原通过皮肤中的树突状细胞（DC）诱导IL-1？mRNA表达，因此我们通过工程XS106来表达皮肤DC系列以表达黄色荧光蛋白（YFP）基因在IL-1促销员的控制下，开发了与HTP兼容的DC生物传感器克隆。我们的目标是开发由KC，DCS和成纤维细胞（FBS）组成的3D多通道皮肤生物传感器系统，并可以简单地检测到皮肤刺激物（通过ATP释放）和皮肤传感器（通过荧光信号测量）。在AIM 1下，我们将通过设计PAM 212 KC线来构建KC生物传感器克隆，以在NRF2的控制下表达GFP基因（一种能够触发keep1-NRF2-ARE细胞对环境应力的细胞存活响应的转录因子）。我们还将生成第二个DC生物传感器克隆，该克隆在TFEC的控制下表达DSRED基因（已知调节细胞因子和细胞因子受体基因表达的转录因子）。 Under Aim 2, we will reconstitute a 3D skin model by: a) cultural the NS FB line in hydrated collagen gel to form a dermal-equivalent matrix, b) cultural 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.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个接触传感器，以评估灵敏度和特异性。我们将测量ATP释放，以指示皮肤刺激性电位和YFP，GFP和DSRED信号，以识别甚至对皮肤传感器进行分类。最后，我们将通过以96个固定板格式重构3D皮肤等效的微文化来提高HTP能力。一旦开发和验证，具有双重功能的3D皮肤模型可能使该行业能够消除动物在皮肤毒理学研究中的使用。在由环境化学物质引起的许多疾病中，接触性皮炎是事件的主要疾病。我们将开发一个三维的多细胞皮肤培养系统，以测试大量工业和环境化学物质，以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皮肤模型可以使该行业消除动物在皮肤毒理学研究中的使用。