Immuno-CometChip for Human Skin Basal Cell Genotoxicity Testing

 描述(由申请人提供)：这项第一阶段STTR研究的目标是量化器官型培养(Epiderm)中的基底细胞角质形成细胞对常用化学试剂的遗传毒性。建议的产品是彗星芯片检测，它测量来自重建的人类表皮的基底细胞的DNA损伤。将解决的技术问题是：1)我们能否修改我们目前使用的彗星芯片分析方法，以加入细胞外基质蛋白或抗体？2)我们能否根据单个基本角质形成细胞与这些基质蛋白(包括I型和IV型胶原)的优先粘附性，或通过使用固定的整合素β1抗体，从3D器官型皮肤培养物中分离单个角质形成细胞？3)我们能否通过针对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？4)我们能否使用分离的、抗体标记的表皮基底细胞检测和量化DNA损伤水平，以响应已知的环境基因毒性物质？5)我们是否可以使用我们的免疫彗星芯片分析方法来筛选目前或计划上市的大量药物？拟议研究的影响将是减少用于毒剂筛选的动物模型，因为人类器官型培养已被证明在其对腐蚀性或刺激性物质的反应方面与人类皮肤的细胞因子谱几乎相同。筛选皮肤遗传毒剂的市场是巨大的，因为目前的筛查程序跟不上目前引入的新药物的数量。目的1建立一种分离基底角质形成细胞的新方法，并建立一种可同时显示β-1整合素和DNA损伤等特异性抗原的免疫染色方法。目标2将使用包括过氧化氢在内的已知DNA损伤剂来验证免疫彗星芯片检测。这为彗星分析增加了三个新参数。第一个是从单个芯片上的器官型培养获得由240个单个基底表皮角质形成细胞组成的96个处理组，第二个是验证它们与表面标志的同一性，第三个是DNA损伤的同时可重复性检测。

 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.