REGULATION OF CYTOCHROME P-450B/E IN HEPATOCYTE CULTURES

肝细胞培养物中细胞色素 P-450B/E 的调节

• 批准号: 3252727
• 负责人: ERIN G SCHUETZ
• 金额: $ 11.07万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-03-01 至 1993-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3252727
• 关键词: DNA; DNA methylation; basement membrane; binding proteins; chemical binding; complementary DNA; cytochrome P450; extracellular matrix; gel electrophoresis; gene expression; genetic manipulation; genetic mapping; genetic transcription; growth media; laboratory mouse; laboratory rat; liver cells; messenger RNA; molecular cloning; morphology; nucleic acid hybridization; nucleic acid probes; nucleic acid sequence; phenobarbital; protein biosynthesis; tissue /cell culture; transferase

The overall objective of this study is to elucidate the controls regulating expression of rat liver cytochromes P450b and P450e (collectively referred to as P450b/e), a gene family of microsomal hemoproteins that catalyze the oxidative metabolism of many lipophilic chemicals. Despite the determined efforts of many laboratories no one has been able to demonstrate the mechanism by which P450b/e gene transcription is increased by a heterogeneous collection of drugs, e.g., phenobarbital (PB), pesticides and lipophilic chemicals. Studies have been hampered by the lack of a cell culture system that retains P450b/e gene expression. My studies are based on the fact that I have discovered a way to preserve cytochrome P450b/e gene expression in rat hepatocytes by culturing them on a basement membrane extract "matrigel". Using specific antibodies and cDNAs as molecular probes, I will first define whether transcriptional or post-transcriptional mechanism are involved in increasing the accumulation of P450b/e mRNA and proteins in response to PB in rat and mouse hepatocytes cultured on matrigel. Second, I will define the extracellular regulatory factors that allow expression of P450b/e in hepatocytes cultured on matrigel by determining whether the effect of matrigel is a function of its composition, the physical state of the matrix or a consequence of its effect on hepatocyte cell shape. The third approach is to identify DNA regulatory sequences within the 5' flanking region of the P450e gene. Established techniques will be used to identify DNAse hypersensitive sites, protein-DNA binding domains and regulatory elements of the P450b/e gene. Finally, I will determine whether these putative DNA regulatory sequences contribute to P450e expression in vivo by transfecting recombinant P450e genes (already formed by cloning progressive deletion fragments of the putative regulatory region of the P450e gene upstream of the reporter gene chloramphenicol acetyltransferase) by electroporation into cultured hepatocytes, treating with various inducers and examining CAT activity. This study will provide a better understanding of liver cell physiology and how the extracellular matrix signals intracellular gene expression. Moreover, this study will provide new and important information on regulation of P450b/e by bringing together for the first time the recombinant P450e plasmids, a P450b/e responsive cell culture system, and a suitable method for introducting biologically active DNA into hepatocytes (electroporation).

本研究的总体目标是阐明 调节大鼠肝细胞色素P450 b和P450 e的表达 （统称为P450 b/e），微粒体基因家族 血红素蛋白，催化许多 亲脂性化学物质。 尽管许多人做出了坚定的努力 实验室里还没有人能够证明这种机制 P450 b/e基因转录增加， 药物的异质集合，例如，苯巴比妥（PB）， 杀虫剂和亲脂性化学品。 研究受到阻碍 由于缺乏保留P450 b/e基因的细胞培养系统， 表情我的研究是基于这样一个事实 发现了一种保存细胞色素P450 b/e基因表达的方法 通过在基底膜上培养大鼠肝细胞， 提取“基质胶”。 使用特异性抗体和cDNA作为 分子探针，我将首先定义是否转录或 转录后机制参与增加 P450 b/e mRNA和蛋白在PB反应中的积累 在基质胶上培养的大鼠和小鼠肝细胞。 第二，我会 定义允许表达的细胞外调节因子 通过测定基质胶上培养的肝细胞中P450 b/e的含量 基质胶的效果是否是其组成的函数， 基质的物理状态或其影响的结果 肝细胞形状。 第三种方法是鉴定DNA P450 e的5'侧翼区域内的调节序列 基因 将使用已建立的技术鉴定DNA酶 超敏位点、蛋白质-DNA结合结构域和调节 P450 b/e基因的突变。 最后，我将决定是否 这些推定的DNA调节序列有助于P450 e 重组P450 e基因的体内表达 （已经通过克隆 P450 e基因上游的推定调控区， 报告基因氯霉素乙酰转移酶） 电穿孔到培养的肝细胞中，用各种 诱导剂和检查CAT活性。 这项研究将提供一个 更好地了解肝细胞生理学以及 细胞外基质信号细胞内基因表达。 而且，这项研究将提供新的重要信息 关于P450 b/e的调节， 重组P450 e质粒，P450 b/e应答细胞 培养系统，以及用于引入 生物活性DNA进入肝细胞（电穿孔）。