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（由申请人提供）：该第一阶段 STTR 研究的目的是量化来自器官型培养物（表皮）的基底细胞角质形成细胞对常用化学试剂的遗传毒性。拟议的产品是彗星芯片检测，可测量源自重建人类表皮的基底细胞中的 DNA 损伤。将解决的技术问题是 1) 我们能否修改目前使用的彗星芯片测定以纳入细胞外基质蛋白或抗体？ 2) 我们能否根据基底角质形成细胞对这些基质蛋白（包括胶原蛋白 I 和 IV）的优先粘附，或通过使用整合素 β1 的固定化抗体，从 3D 器官型皮肤培养物中分离出单个基底角质形成细胞？ 3）我们可以通过针对α2或β1（胶原配体）、整合素的量子点偶联抗体来确认它们的身份吗？ 4) 我们能否使用分离的、抗体标记的表皮基底细胞来检测和量化已知环境基因毒性剂引起的 DNA 损伤水平？ 5) 我们可以使用我们的免疫彗星芯片检测来筛选目前或拟上市的大量药物吗？拟议研究的影响将是减少用于有毒物质筛选的动物模型，因为人类器官培养物已被证明在对腐蚀性或刺激性物质的细胞因子谱方面几乎与人类皮肤相同。由于目前的筛选程序无法跟上目前推出的新试剂的数量，因此筛查皮肤遗传毒性试剂的市场巨大。目标 1 是开发一种分离基底角质形成细胞的新方法，以及一种同时可视化特定抗原（包括 β1 整合素和 DNA 损伤）的免疫染色方法。目标 2 是使用已知的 DNA 损伤剂（包括 H2O2）验证免疫彗星芯片测定。这为彗星测定增加了三个新参数。第一个是从单个芯片上的器官型培养物中获得 96 个治疗组，每组 240 个单个基底表皮角质形成细胞，第二个是用表面标记验证它们的身份，第三个是同时可重复的 DNA 损伤测定。