FUNCTION AND REGULATION OF GENE EXPRESSION IN LUNG INJURY

肺损伤中基因表达的功能和调控

• 批准号: 5213595
• 负责人: STUART HOROWITZ
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5213595
• 关键词: DNA binding protein; cytokine; extracellular matrix proteins; gene expression; gene induction /repression; genetic regulation; growth factor; hyperoxia; immunocytochemistry; in situ hybridization; laboratory mouse; lung injury; mature animal; messenger RNA; metallothionein; molecular cloning; molecular pathology; newborn animals; nucleic acid sequence; protease inhibitor; pulmonary fibrosis /granuloma; radiobiology; tissue /cell culture; tissue inhibitor of metalloproteinases; transcription factor

The overall goal of these studies is to improve our understanding of the biochemical basis of the lung's response to acute injury. Toward this end, this proposal seeks to elucidate the mechanisms of regulation and function of genes that have been found to exhibit notable increases in their mRNA levels in the lung after exposure to hyperoxia and administration of oleic acid, as models of acute injury. The unifying concept underlying these studies is that there may be similarities in the mechanisms that activate these genes. The rationale is that once these mechanisms are understood they may then be controlled, allowing mitigation of the lung injury. The first Specific Aim is to compare the cell-specific accumulation of lung injury-related (LIR) mRNAs during the acute phase of hyperoxic lung injury in adult and newborn rabbits, and in rabbits treated intravenously with oleic acid. In situ hybridization and immunocytochemistry will be used to test the hypothesis that LIR mRNAs are expressed in a temporally-and spatially-specific manner in cells of the injured lung, depending on the extent of localized injury. The second Specific Aim is to isolate and characterize genomic clones for rabbit genes encoding the tissue inhibitor of metalloproteinases (TIMP) and metallothionein (MT), two LIR genes. The ends of their mRNAs will also be mapped. Comparisons of the putative regulatory portions of these genes will begin to test the hypothesis that there are shared and distinct mechanisms of their induction during lung injury. These data are also essential prerequisites to later studies of the mechanisms of their regulation. The third Specific Aim is to understand the function of metallothioneins in acute lung injury. Using stable and transient gene expression systems which are currently available, two hypothetical functions of MT will be tested. The fourth Specific Aim is to study the effects of oxygen, and polypeptide growth factors and cytokines as molecular mediators of TIMP and MT induction. The isolated, perfused, and ventilated lung model will be used as a bridge, to close the gap between previous in vivo studies and additional experiments of this aim, in cultured pulmonary endothelial cells and fibroblasts, to identify the molecular mediators of LIR gene induction. The fifth Specific Aim is to elucidate the molecular mechanisms underlying the increase in TIMP and MT mRNAs in pulmonary endothelial cells and fibroblasts, respectively, during acute lung injury. In vitro DNA-protein binding and reciprocal competition experiments will more directly test the hypothesis that there are unique and overlapping mechanisms of differential and coordinate LIR gene induction.

这些研究的总体目标是提高我们对 肺对急性损伤的反应的生化基础。 朝着这一目标， 该提案旨在阐明调节和功能的机制 发现其mRNA表现出显着增加的基因 暴露于高氧和给药的肺部水平 酸，作为急性损伤的模型。 这些统一的概念 研究是激活的机制可能存在相似之处 这些基因。 理由是一旦理解了这些机制 然后可以控制它们，从而减轻肺损伤。 这 第一个具体目的是比较肺的细胞特异性积累 在高氧肺损伤的急性阶段与损伤相关（LIR）mRNA 在成年和新生兔子中，以及静脉注射的兔子 油酸。 原位杂交和免疫细胞化学将用于 检验LIR mRNA在时间上表达的假设 受伤肺细胞中的空间特异性方式，取决于 局部伤害的程度。 第二个具体目的是隔离和 表征编码组织抑制剂的兔基因的基因组克隆 金属蛋白酶（TIMP）和金属硫蛋白（MT），两个LIR基因。 这 他们的mRNA的末端也将被映射。 假定的比较 这些基因的调节部分将开始检验以下假设。 在肺部诱导的共享机制和不同的机制 受伤。 这些数据也是以后研究的必要前提 其调节的机制。 第三个具体目的是 了解金属霉素在急性肺损伤中的功能。 使用 当前可用的稳定和瞬态基因表达系统 将测试MT的两个假设功能。 第四个特定目标 是研究氧气和多肽生长因子以及 细胞因子作为TIMP和MT诱导的分子介质。 孤立的， 灌注和通风的肺模型将用作桥梁，以关闭 以前的体内研究与此其他实验之间的差距 目的是在培养的肺内皮细胞和成纤维细胞中识别 LIR基因诱导的分子介体。 第五个具体目标是 阐明TIMP和 肺内皮细胞和成纤维细胞中的mt mRNA， 在急性肺损伤期间。 体外DNA-蛋白结合和倒数 竞争实验将更直接地检验其中的假设 是差分和坐标LIR的独特而重叠的机制 基因诱导。