PROTEIN SYNTHESIS IN NORMAL AND REGENERATING LIVER

正常肝脏和再生肝脏中的蛋白质合成

• 批准号: 3225798
• 负责人: DAVID A SHAFRITZ
• 金额: $ 24.87万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-05-01 至 1989-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3225798
• 关键词: Escherichia coli; cell differentiation; cell free system; density gradient ultracentrifugation; developmental genetics; electrofocusing; electron microscopy; electrophoresis; eukaryote; gel filtration chromatography; gene expression; genetic manipulation; genetic recombination; genetic transcription; genetic translation; hepatocellular carcinoma; immunochemistry; liver cells; liver cirrhosis; liver metabolism; liver regeneration; liver toxic disorder; messenger RNA; molecular cloning; mutant; nucleic acid sequence; radiotracer; renal failure; tissue /cell culture

Studies on mechanisms regulating hepatic protein synthesis have evolved into analysis of liver-specific gene function using molecular hybridization and cell-free protein synthesis. We have developed in situ hybridization to detect mRNAs of high (albumin) and low (proalpha2-collagen) abundance in individual hepatocytes, in vitro transcription with isolated nuclei to directly measure transcription rates for liver-specific genes under various experimental conditions and transfection methods for expressing foreign genes in primary rat hepatocytes and hepatoma cells. Our current specific aims are (1) to study the mechanism regulating transcription and processing of albumin mRNA in normal rat liver and an analbuminemic rat model in which abnormal albumin mRNA processing is thought to occur, (2) to study the role of an acute hepatotoxic agent (CCl4) on transcription of liver-specific versus general cellular genes, (3) to determine whether chronic CCl4 administration induces hepatocytes to synthesize collagen, and whether such induction participates in development of hepatic fibrosis, (4) to study regulation of specific gene transcription in developing and regenerating liver, and (5) to develop a method for introducing molecularly engineered plasmid vectors containing specific genes of interest into hepatocytes and/or hepatoma cells. Specific genes to be studied include albumin, Alpha-fetoprotein, apolipoprotein A1 and E, Alpha1-antitrypsin (secreted proteins synthesized in liver), ligandin and cytochrome P-450 (intracellular proteins enriched in liver), collagen, Beta-actin and Alpha-tubulin (genes of general cellular function), and Beta-globin and pBR322 as negative controls. In preliminary studies, we have observed immediate reduction in albumin gene transcription following administration of a single dose of CCl4, followed by a sharp increase in Alpha-fetoprotein transcription, and later, increased albumin transcription as the liver regenerates. In analbuminemic rats, albumin transcription responds normally to acute CCl4 administration; however, Alpha-fetoprotein transcription does not increase. This model will be used to study factors regulating transcription of these two genes in coordinate or non-coordinate fashion. Collagen gene expression and specific cells in the liver containing collagen mRNA during development of hepatic fibrosis and the role of corticosteroids in ameliorating this response will be studied by in situ hybridization together with collagen synthesis measurements. The role of cis or transacting factors in regulating albumin and Alpha-fetoprotein expression will be studied by introducing plasmid expression vectors containing these genes and/or upstream regulatory elements into hepatocytes and/or hepatoma cells in culture. Finally, attempts will be made to introduce biologically active albumin genes into analbuminemic rat hepatocytes to obtain stable expression of these genes and if clones become available, to perform similar studies for UDPGT-deficiency in the Gunn rat.

肝脏蛋白质合成调控机制的研究进展 分子杂交法分析肝脏特异性基因功能 和无细胞蛋白质合成。我们已经开发出原位杂交技术 检测高(白蛋白)和低(前α2-胶原)丰度的mRNAs 单个肝细胞，用分离的核进行体外转录 直接测量肝脏特异基因在不同环境下的转录速率 外源基因表达的实验条件和转染方法 原代大鼠肝细胞和肝癌细胞中的基因。我们目前的具体情况 目的是(1)研究转录和加工的调控机制 正常大鼠肝脏白蛋白mRNA的表达及大鼠蛋白缺乏症模型 白蛋白mRNA的异常加工被认为是发生的，(2)研究其作用 急性肝毒剂(CCl4)对肝脏特异性转录的影响 与一般细胞基因相比，(3)确定慢性CCl4 给药诱导肝细胞合成胶原，以及是否如此 诱导参与肝纤维化的发展，(4)研究 发育和再生过程中特定基因转录的调控 肝脏，以及(5)开发一种引入分子工程的方法 含有特定目的基因的肝细胞表达载体 和/或肝癌细胞。待研究的特定基因包括白蛋白， 甲胎蛋白、载脂蛋白A1和E、甲胎蛋白抗胰蛋白酶(分泌型 肝脏合成的蛋白质)、配基和细胞色素P-450 (细胞内蛋白在肝脏中富含)、胶原、β-肌动蛋白和 α-微管蛋白(一般细胞功能基因)和β-珠蛋白和 PBR322作为阴性对照。在初步研究中，我们观察到 白蛋白基因转录在给药后立即降低 单剂CCl4，随后甲胎蛋白急剧增加 转录，以及后来，随着肝脏白蛋白转录的增加 再生。在白蛋白血症大鼠中，白蛋白转录反应 正常到急性给药；然而，甲胎蛋白 转录不会增加。这个模型将被用来研究影响因素 协同或非协同调控这两个基因的转录 时尚。肝组织中胶原基因的表达和特异性细胞 肝纤维化发生发展过程中胶原基因的表达及意义 皮质类固醇在改善这种反应中的作用将由英国医学学会研究。 原位杂交结合胶原合成测定。角色 调节白蛋白和甲胎蛋白的顺式或交易因子 将通过引入表达载体来研究表达 将这些基因和/或上游调控元件导入肝细胞 和/或培养中的肝癌细胞。最后，我们将尝试 将具有生物活性的白蛋白基因导入高蛋白血症大鼠 肝细胞获得这些基因的稳定表达，如果克隆成为 可用于对Gunn大鼠的UDPGT缺乏进行类似的研究。