Definition of the mechanisms that underlie the decrease in expression of the cytochrome P450s in primary hepatocytes

原代肝细胞中细胞色素 P450 表达减少的机制的定义

• 批准号: BB/E010040/1
• 负责人: Christopher Goldring
• 金额: $ 29.53万
• 依托单位: University of Liverpool
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2007
• 资助国家: 英国
• 起止时间: 2007 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FE010040%2F1
• 关键词: Definition; mechanisms; underlie; decrease; expression

A physiological understanding of how biological systems respond to xenobiotics and endobiotics is fundamental to the prediction of the consequences of exposure to dietary, environmental, and pharmaceutical products. Currently, the investigation of mammalian responses to these products is reliant on whole animal studies, partly due to the lack of reliable, stable and cost effective in vitro alternatives that faithfully reflect their disposition in an integrated physiological system. This is particularly true for metabolism studies where isolation and/or culture of liver cells leads to de-differentiation and loss of multiple enzyme functions. The ideal in vitro model for understanding the physiological consequences of exposure of the liver to drugs and chemicals is a metabolically-competent hepatocyte. To be a physiologically adequate model, hepatocytes should contain a normal complement of the cytochrome P450 proteins (CYP450s) essential for the chemical modification and clearance of the majority of foreign compounds, as well as many endogenously-formed compounds. Unfortunately, expression of most of the relevant CYP450s in freshly isolated hepatocytes is lost within days of their transfer into laboratory culture conditions. The loss of the CYP450s from these cells is associated with a reduction in the activity of a number of regulatory proteins. However, the identity of all the transcription factors contributing to the maintenance of the CYP450s in primary cells and the extent to which these determine the basal level of CYP450s in hepatocytes is not known. Therefore, the main question that I wish to address in this proposal is what are the molecular mechanisms that determine the decrease in expression of the CYP450s in primary hepatocytes in culture? In parallel with this question, I will attempt to test the hypothesis that the manipulation of positive and/or negative regulators of CYP450 expression in primary hepatocytes can yield a more physiologically relevant Phase I phenotype. A twin-track approach is proposed in this application that aims to help determine the events that occur during the loss of the CYP450s from hepatocytes in culture. This will consist of a measurement of the principal candidate transcriptional regulators in a primary hepatocyte model, alongside an unbiased global screen of nuclear transcription factor changes that accompany the classical loss of the Phase 1 metabolic phenotype in these cells. The technology that will be employed in this study benefits from the great advances in gene transfection and RNA interference in primary cells that have occurred only recently. This will allow the manipulation of the regulatory factors that are implicated in the control of the CYP450s, and represents a fresh approach to tackle this important question.

从生理学角度理解生物系统对外源性和内源性药物的反应是预测暴露于饮食、环境和药物产品的后果的基础。目前，哺乳动物对这些产品反应的研究依赖于全动物研究，部分原因是缺乏可靠、稳定和具有成本效益的体外替代品，无法忠实地反映它们在综合生理系统中的分布。在代谢研究中尤其如此，因为肝细胞的分离和/或培养会导致去分化和多种酶功能的丧失。了解肝脏暴露于药物和化学物质的生理后果的理想体外模型是具有代谢能力的肝细胞。为了成为一个生理上适当的模型，肝细胞应该含有正常的细胞色素P450蛋白（cyp450）补体，这对于大多数外来化合物以及许多内源性形成的化合物的化学修饰和清除至关重要。不幸的是，在新鲜分离的肝细胞中，大多数相关cyp450的表达在转移到实验室培养条件后的几天内就消失了。这些细胞中cyp450的缺失与一些调节蛋白活性的降低有关。然而，参与原代细胞中cyp450维持的所有转录因子的身份，以及这些转录因子在多大程度上决定肝细胞中cyp450的基础水平，目前尚不清楚。因此，我希望在这个提案中解决的主要问题是，在培养的原代肝细胞中，决定cyp450表达减少的分子机制是什么？与这个问题并行，我将尝试验证这样一个假设，即在原代肝细胞中操纵CYP450表达的阳性和/或阴性调节因子可以产生更生理相关的I期表型。在本应用中提出了一种双轨方法，旨在帮助确定培养肝细胞中cyp450丢失期间发生的事件。这将包括原发性肝细胞模型中主要候选转录调节因子的测量，以及这些细胞中伴随经典1期代谢表型丧失的核转录因子变化的无偏性全球筛选。这项研究将采用的技术得益于最近在原代细胞中基因转染和RNA干扰方面取得的巨大进展。这将允许操纵与cyp450控制有关的调节因子，并代表了解决这一重要问题的新方法。

项目成果

Network Analysis of Primary Hepatocyte Dedifferentiation Using a Shotgun Proteomics Approach

使用鸟枪蛋白质组学方法进行原代肝细胞去分化的网络分析

• DOI: 10.1021/pr1001687
• 发表时间: 2010
• 期刊: Journal of Proteome Research
• 影响因子: 4.4
• 作者: Rowe C

Generating hepatic cell lineages from pluripotent stem cells for drug toxicity screening.

• DOI: 10.1016/j.scr.2010.02.002
• 发表时间: 2010-07
• 期刊: STEM CELL RESEARCH
• 影响因子: 1.2
• 作者: Baxter, Melissa A.;Rowe, Cliff;Alder, Jane;Harrison, Sean;Hanley, Karen Piper;Park, B. Kevin;Kitteringham, Neil R.;Goldring, Chris E.;Hanley, Neil A.
• 通讯作者: Hanley, Neil A.

Role of protein haptenation in triggering maturation events in the dendritic cell surrogate cell line THP-1.

蛋白质半抗原化在触发树突状细胞替代细胞系 THP-1 成熟事件中的作用。

• DOI: 10.1016/j.taap.2009.05.001
• 发表时间: 2009
• 期刊: Toxicology and applied pharmacology
• 影响因子: 3.8
• 作者: Megherbi R