Measuring toxicant effects on cellular function in a microarray format

项目摘要/摘要-美国环保署保持了一个有毒化学品释放清单> 650有毒化学品， 美国有超过20，000个工业场所处理或释放，而HHS和USDA保持着一份清单， 超过60种对人类、动物和植物健康构成严重风险的特定物质和有毒物质。这些 这些化合物仅代表了已知和未知的环境毒物的一小部分， 影响人类健康。新的和未知的有毒物质以前所未有的速度合成， 这些物质的数量正在迅速进入环境。因此，迫切需要一种高含量的测定方法， 有毒物质对细胞功能的影响这样一个平台将扩大我们对环境的理解 可能代表自身免疫性疾病、免疫缺陷和瘤形成风险的成分。 干细胞是胚胎干细胞的重要组成部分，是毒物的重要和敏感的靶标。 发育和维持成人组织是不可或缺的。测量股骨柄变化的当前试验 细胞分化和编程反应的毒性作用提供了深入了解的发病机制， 有毒物质暴露，但这些技术不能够保持快速的速度所需的吞吐量 新的有毒物质进入环境。此外，执行劳动密集型金- 标准测试限制了收集的细胞群体、毒物身份和暴露的数据量 浓度.尽管如此，这些测试是定期进行的，因为这些测量对我们的 了解这些物质所代表的风险，以及我们减轻或消除这些风险的能力。 本项目的目标是开发能够检测干细胞毒性作用的仪器 在敏感的高含量测定中的分化和行为。本试验将基于以下检测： 空间编码微阵列的表面标志物表达和细胞因子分泌谱的变化， 干细胞这些细胞将同时暴露于一个或多个毒物浓度梯度， 将代表暴露或两种试剂共同暴露的光谱，这些暴露或两种试剂共同暴露可能由细胞在 环境科学家们正在提出一种仪器，可以测量光栅耦合表面等离子体， 共振以及来自三种不同荧光团的等离子体增强的荧光发射。这 该系统还将包括一个变焦透镜，它允许全芯片成像和高放大率的单点成像 图像.它将支持芯片上孵育和允许细胞暴露于扩散的样品室。 毒性梯度，使其成为理想的实验室评价毒性对干细胞分化的影响。 还可以同时评估对其他细胞谱系的影响。该提议的仪器 将是高度通用的，随着项目的进展， 第三阶段。这种性质的经过充分验证的平台可能很容易适用于药物发现工作， 化合物验证，并可能最终发现作为临床诊断的价值。

Project Summary/Abstract – The EPA maintains a Toxics Release Inventory of >650 toxic chemicals that are disposed or released from >20,000 industrial sites in the United States, while HHS and USDA maintain a list of over 60 select agents and toxicants that pose a severe risk to human, animal, and plant health. These compounds represent only a fraction of the known and unknown environmental toxicants that may adversely affect human health. New and uncharacterized toxicants are synthesized at an unprecedented rate, and trace quantities of these are rapidly entering the environment. There is thus a critical need for a high-content assay of the effects of toxicants on cell function. Such a platform would broaden our understanding of environmental components that may represent risks for autoimmune disease, immunodeficiencies, and neoplasia. Stem cells are an important and sensitive target for toxicants as they are critical components of embryonic development and are integral to the maintenance of adult tissues. Current assays measuring changes in stem cell differentiation and programming in response to toxic effects provide insight into the pathogenesis of toxicant exposure, but these techniques are not capable of the throughput necessary to stay apace the speed of new toxicants entering the environment. Further, the expense of performing the labor-intensive gold- standard tests limits the amount of data gathered for a cell population, toxicant identity, and exposure concentration. Nonetheless, these tests are regularly performed since such measurements are critical to our understanding of the risks these agents represent, and to our ability to moderate or eliminate those risks. The goal of this project is the development of instrumentation capable of detecting toxicant effects on stem cell differentiation and behavior in a sensitive high-content assay. This assay will be based on the detection of changes in the surface marker expression and cytokine excretion profile of a spatially encoded microarray of stem cells. These cells will be exposed to one or more simultaneous toxicant concentration gradients, which will represent the spectrum of exposures or two-agent co-exposures that might be encountered by cells in the environment. Ciencia is proposing instrumentation that can measure both grating-coupled surface plasmon resonance as well as plasmonically-enhanced fluorescent emission from three distinct fluorophores. This system will also incorporate a zoom lens that permits whole-chip imaging and high-magnification single-spot images. It will support on-chip incubation and a sample chamber that permits exposure of cells to a diffusing gradient of toxicant, making it ideal for the laboratory evaluation of toxicant effects on stem cell differentiation. Concurrent assessments of effects on other cell lineages may also performed. This proposed instrumentation would be highly versatile, with modular components suggesting additional utility as the project progresses to Phase III. A well-validated platform of this nature may be readily applicable to drug discovery efforts and lead compound validation, and may ultimately find value as a clinical diagnostic.