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

这项第一阶段的提案将开发和验证测量化合物新陈代谢的分析方法。 以及它们对肝细胞的影响 保护试验(QNAP)，可以测量高达100个mRNA和miRNA的表达，是一种 简单、可靠和高精度的分析，提供高样品吞吐量。两个蜂窝系统将是 经鉴定，HepaRG细胞系和原代肝细胞。与新陈代谢相关的基因是已知的， 他们将被纳入化验中。与新陈代谢相关的miRNA是未知的，它们 将使用完整的转录组miRNA分析进行鉴定。最终的筛查化验将包括这两项内容 MRNA和miRNA，并将以384井的形式进行。生成的数据将是剂量响应 数据，以便可以根据受调控的基因和EC50来评估差异代谢 他们在那里受到监管。 重要的是，这种分析方法将能够筛选出在细胞系统中代谢的化合物。 这反映了体内的肝功能，并评估了化合物代谢的所有途径， 包括一期和二期代谢，以及转运体，这样不仅初级化合物会 影响将被监测，但也将评估细胞系统产生的代谢物的影响。这 将在一次化验中完成，这是酶分析无法完成的。获得剂量 反应数据和EC50的S在基因表达水平上具有很高的新颖性，并将基因表达提高到 今天新陈代谢酶检测的水平。

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.