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: 3252731
• 负责人: ERIN G SCHUETZ
• 金额: $ 14.55万
• 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/3252731
• 关键词: DNA; DNA methylation; basement membrane; binding proteins; chemical binding; complementary DNA; cytochrome P450; extracellular matrix; gel electrophoresis; gene expression; gene induction /repression; 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控制措施 调节大鼠肝细胞色素P450b和P450e的表达 （统称为P450b/e），微粒体基因家族 血红素蛋白可催化多种物质的氧化代谢 亲脂性化学物质。 尽管许多人付出了坚定的努力 实验室尚未有人能够证明这一机制 P450b/e 基因转录增加 药物的异质集合，例如苯巴比妥 (PB)、 农药和亲脂性化学品。 研究受到阻碍 由于缺乏保留 P450b/e 基因的细胞培养系统 表达。我的研究基于以下事实： 发现了一种保留细胞色素 P450b/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，我将首先定义转录或 转录后机制参与增加 PB 反应中 P450b/e mRNA 和蛋白质的积累 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 通过测定在基质胶上培养的肝细胞中的 P450b/e 基质胶的效果是否与其成分有关， 基质的物理状态或其影响的结果 肝细胞的细胞形状。 第三种方法是鉴定DNA P450e 5' 侧翼区域内的调控序列 基因。 已建立的技术将用于识别 DNAse 超敏感位点、蛋白质-DNA 结合域和调控 P450b/e 基因的元件。 最后我会确定是否 这些假定的 DNA 调控序列有助于 P450e 通过转染重组 P450e 基因进行体内表达 （已经通过克隆渐进删除片段形成 P450e 基因上游的假定调控区 报告基因氯霉素乙酰转移酶） 电穿孔到培养的肝细胞中，用各种处理 诱导剂并检查 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如何 细胞外基质向细胞内基因表达发出信号。 此外，这项研究将提供新的重要信息 首次汇集P450b/e的监管 重组 P450e 质粒，P450b/e 反应细胞 培养系统以及合适的引入方法 将生物活性 DNA 导入肝细胞（电穿孔）。