TAS::75 0862::TAS SBIR 2010 TOPIC 109

塔斯马尼亚州::75 0862::塔斯马尼亚州 SBIR 2010 主题 109

• 批准号: 8163995
• 负责人: KIRK COLLAMER
• 金额: $ 14.94万
• 依托单位: HTG MOLECULAR DIAGNOSTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-30 至 2011-06-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8163995
• 关键词: Affect; Automation; Biological Assay; Cell Line; Cells; Companions; Cytolysis; Data; Dimethyl Sulfoxide; Dose; Enzymes; Gene Expression; Gene Expression Profile; Genes; Hepatocyte; Maps; Measures; Messenger RNA; Metabolism; MicroRNAs; Monitor; Nuclease Protection Assays; Pathway interactions; Phase; Practice Guidelines; Reporting; Reproducibility; Research; Sampling; Screening procedure; Services; Small Business Innovation Research Grant; System; Testing; Time; Validation; base; blind; cell type; cost effective; in vivo; liver function; novel; response

This Phase I proposal will develop and validate assays to measure the metabolism of compounds and their affect on liver cells using a multiplexed gene expression assay platform, the quantitative Nuclease Protection Assay (qNAP¿) that can measure the expression of up to 100 mRNA and miRNA and is a simple, robust and highly precise assay that provides high sample throughput. Two cell systems will be evaluated, HepaRG cell line and primary hepatocytes. The genes associated with metabolism are known, and they will be incorporated into the assay. miRNA associated with metabolism are not known, and they will be identified using a whole transcriptome miRNA assay. The final screening assay will incorporate both mRNA and miRNA, and will be performed in a 384-well format. The data generated will be dose response data so that differential metabolism can be assessed based of what genes are regulated as well as the EC50 at which they are regulated. The significance is that this assay will enable the screening of compounds for metabolism on a cell system that mirrors in vivo liver function and assess all the pathways by which compounds are metabolized, including Phase I and Phase II metabolism, and transporters, so that not only will the primary compound effects be monitored, but also the effect of metabolites produced by the cell system will be assessed. This will be done ins a single assay, something that cannot be done with enzyme assays. Acquiring dose response data and EC50's at the level of gene expression is highly novel, and brings gene expression up to the level where metabolism enzyme assays are today.

该第一阶段提案将开发和验证测定化合物代谢的方法 以及它们对肝细胞的影响，使用多重基因表达测定平台，定量核酸酶 保护试验（qNAP），可以测量多达100种mRNA和miRNA的表达， 提供高样品通量的简单、稳健和高度精确的测定。两个细胞系统将是 评价了HepaRG细胞系和原代肝细胞。与新陈代谢相关的基因是已知的， 并且它们将被结合到测定中。与代谢相关的miRNA尚不清楚，它们 将使用全转录组miRNA测定来鉴定。最终筛选试验将包括 mRNA和miRNA，并将在384孔格式中进行。生成的数据将是剂量反应 数据，以便可以根据哪些基因受到调控以及EC50来评估差异代谢 在他们被监管的时候。 重要的是，该测定将能够筛选用于细胞系统代谢的化合物 反映体内肝功能并评估化合物代谢的所有途径， 包括I期和II期代谢和转运蛋白，因此不仅主要化合物 除了监测效果外，还将评估细胞系统产生的代谢物的效果。这 将在一次检测中完成，这是酶检测无法完成的。采集剂量 在基因表达水平上的响应数据和EC50是高度新颖的，并且使基因表达达到 代谢酶检测的水平。