Immuno-CometChip for Human Skin Basal Cell Genotoxicity Testing

 描述（由申请方提供）：本1期STTR研究的目的是量化来自器官型培养物（表皮细胞）的基底细胞角质形成细胞对常用化学试剂的遗传毒性反应。申报产品是一种彗星芯片测定法，可测量来自重建人表皮的基底细胞中的DNA损伤。将解决的技术问题是1）我们能否修改我们目前使用的彗星芯片测定法，以纳入细胞外基质蛋白或抗体？2)我们是否可以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单个基底角质形成细胞？3)我们能否通过对α2或β1（胶原配体）、整合素特异性的量子点偶联抗体来确认它们的身份？4)我们是否可以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遗传毒性剂引起的DNA损伤水平？5)我们是否可以使用我们的免疫彗星芯片检测来筛选大量目前或拟上市的药物？拟议研究的影响将是减少用于毒性剂筛选的动物模型，因为人类器官型培养物已被证明与人类皮肤在对腐蚀性或刺激性剂的反应中的细胞因子谱几乎相同。筛选皮肤遗传毒性剂的市场是巨大的，因为目前的筛选程序不能跟上目前正在引入的新药剂的数量。目的1：建立一种分离基底角朊细胞的新方法，并建立一种能同时显示β1整合素和DNA损伤等特异性抗原的免疫染色方法。目标2将是使用已知的DNA损伤剂（包括H2O2）验证免疫彗星芯片检测。这为彗星试验增加了三个新参数。第一个是从单个芯片上的器官型培养物获得96个治疗组的240个单个基底表皮角质形成细胞，第二个是用表面标志物验证它们的身份，第三个是DNA损伤的同时可重复测定。

 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.