Immuno-CometChip for Human Skin Basal Cell Genotoxicity Testing

用于人体皮肤基底细胞遗传毒性测试的免疫彗星芯片

• 批准号: 9136447
• 负责人: Jay George
• 金额: $ 15.3万
• 依托单位: TREVIGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9136447
• 关键词: Address; Adhesions; Alkalies; Allergic; Animal Model; Antibodies; Antigens; Basal Cell; Binding; Biological Assay; Caliber; Cell Separation; Cells; Chemical Agents; Chemicals; Collagen; Comet Assay; Corrosives; Coupled; Cryopreserved Cell; DNA Damage; Deposition; Epidermis; Extracellular Matrix Proteins; Gel; Goals; Grant; Household; Human; Hydrogen Peroxide; Hypersensitivity skin testing; Imagery; Individual; Integrins; Label; Ligands; Light; Market Research; Marketing; Measures; Methods; Modeling; Monitor; Mutagenicity Tests; Mutagens; New Agents; Peptide Hydrolases; Phase; Process; Proteins; Protocols documentation; Quantum Dots; Research; Screening procedure; Sepharose; Skin; Small Business Technology Transfer Research; Staging; Stem cells; Surface; Techniques; Testing; Time; Toxic effect; Toxicity Tests; Universities; Validation; base; chromatin immunoprecipitation; cold temperature; cytokine; fluorescence microscope; genotoxicity; interest; keratinocyte; nanoparticle; public health relevance; rapid technique; research and development; response; screening; success; treatment group

 DESCRIPTION (provided by applicant): The objective of this Phase 1 STTR research is to quantify genotoxicity in basal cell keratinocytes from organotypic cultures (Epiderm) in response to commonly used chemical agents. The proposed product is a Comet Chip assay that measures DNA damage in basal cells derived from a reconstructed human epidermis. Technical questions that will be addressed are 1) Can we modify our currently used Comet Chip assay to incorporate extracellular matrix proteins or antibodies? 2) Can we isolate individual basal keratinocytes from a 3D organotypic skin culture on the basis of their preferential adhesion to these matrix proteins, including collagen I and IV, or by using immobilized antibodies to integrin β1? 3) Can we confirm their identity by quantum dot-coupled antibodies specific for α2 or β1 (collagen ligand), integrins? 4) Can we use the isolated, antibody-labeled epidermal basal cells to detect and quantify levels of DNA damage in response to known environmental genotoxic agents? 5) Can we use our Immuno-CometChip assay to screen large numbers of agents currently or proposed to be marketed? The impact of the proposed research will be to reduce animal model use for toxic agent screening, since human organotypic culture has been shown to be almost identical to human skin with respect to its cytokine profile in response to corrosive or irritating agents. The market for screening skin genotoxic agents is immense, since the current screening procedures cannot keep pace with the number of new agents currently being introduced. Aim 1 will be to develop a new method for the isolation of basal keratinocytes, and an immunostaining method for simultaneous visualization of specific antigens, including β1 integrin and DNA damage. Aim 2 will be to validate the Immuno-CometChip assay using known DNA damaging agents, including H2O2. This adds three new parameters to the Comet assay. The first is obtaining 96 treatment groups of 240 single basal epidermal keratinocytes from an organotypic culture on a single chip, the second is verifying their identity with surface markers, and the third is the simultaneous reproducible assay for DNA damage.

 描述（由适用提供）：该阶段1 STTR研究的目的是量化有机培养物（Epiderm）基本细胞角质形成细胞中的遗传毒性，以响应常用的化学剂。所提出的产品是一种彗星芯片分析，可测量从重建的人表皮衍生的基本细胞中的DNA损伤。将要解决的技术问题是1）我们是否可以修改当前使用的彗星芯片测定法以结合细胞外基质蛋白或抗体？ 2）我们可以根据对这些基质蛋白的首选粘合（包括胶原蛋白I和IV）或对整合素β1的固定抗体使用基本的有机皮肤培养？ 3）我们可以通过对α2或β1（胶原配体）的量子偶联抗体（整联蛋白）确认其身份？ 4）我们可以使用分离的抗体标记的表皮碱性细胞来检测和量化DNA损伤水平，以响应已知的环境遗传毒性剂？ 5）我们可以使用免疫 - 莫奇基测定法筛选当前的大量代理商或提议进行销售？拟议的研究的影响是减少动物模型对有毒剂筛查的使用，因为人类有机培养在响应腐蚀性或刺激性剂的情况下，就其细胞因子的细胞因子概况而言，人类有机培养几乎与人类皮肤相同。筛选皮肤遗传毒性剂的市场巨大，因为当前的筛选程序无法与当前正在引入的新代理数量保持同步。目标1将是开发一种新方法来分离碱性角质形成细胞，以及一种免疫染色方法，用于简单可视化特定抗原，包括β1整合素和DNA损伤。 AIM 2将是使用已知的DNA损伤剂（包括H2O2）验证免疫 - 莫基测定法​​。这将在彗星测定中添加三个新参数。第一个是从单个芯片上的有机培养物中获得240个单个基本表皮角质形成细胞的96个治疗组，第二个是验证其与表面标记的身份，第三个是对DNA损伤的简单可重复的测定法。