FIBROBLAST ACTIVATION IN URINARY OBSTRUCTION

尿路梗阻中成纤维细胞的激活

• 批准号: 6564088
• 负责人: STEPHEN L GLUCK
• 金额: $ 18.65万
• 依托单位: BARNES-JEWISH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6564088
• 关键词: DNA binding protein; actins; cell differentiation; cellular pathology; crosslink; extracellular matrix proteins; fibroblasts; fibrosis; gel mobility shift assay; gene expression; genetic promoter element; genetic regulation; genetic transcription; green fluorescent proteins; interstitial nephritis; kidney disorder; laboratory mouse; molecular pathology; protein isoforms; renal tubule; smooth muscle; tissue /cell culture; urinary tract obstruction

DESCRIPTION (Abstract of the project) Urinary tract obstruction and other conditions that stimulate renal fibrogenesis induce a change in the state of differentiation of parenchymal fibroblasts characterized by a marked increase in the secretion of several classes of collagen, and the acquisition of a contractile phenotype associated with expression of a-smooth muscle actin: the "myofibroblast." A number of studies in the intact kidney have implicated angiotensin II, TGF-B1, and other agents as key factors required for myofibroblast differentiation. In contrast, studies on a-smooth muscle actin expression in fibroblasts and other cells in culture have yielded conflicting results. Preliminary evidence presented in this application indicates that TGF-BI has little or no effect on the expression of mammalian fibroblasts in culture. The focus of this project will be to identify the DNA regulatory elements and DNA-binding proteins in the a-smooth muscle actin promoter responsible for urinary obstruction-dependent expression in fibroblasts. BALB/c mouse fibroblasts will be marked by stable expression of green fluorescent protein (GFP), and injected into the kidneys of syngeneic mice, where they will be assessed for obstruction-dependent induction of a-smooth muscle actin (SMA) expression. The marked fibroblasts will then be stably transfected with SMA promoter-reporter constructs using the, firefly and Renilla luciferase dual reporter system to allow identification of DNA elements essential for obstruction-dependent expression. Mobility shift assays and other techniques will then be used to identify the DNA-binding proteins interacting with these sites. Finally, the syngeneic fibroblast system will be used to express polyhistidine-tagged proteins in the interstitium of obstructed and contralateral kidneys, to identify the determinants of extracellular matrix accumulation. These studies will attempt to identify factors essential for the initiation of tubulointerstitial fibrosis in urinary tract obstruction. The results may lead to the identification of new therapeutic targets for arresting and reversing interstitial fibrosis.

描述（项目摘要） 尿路阻塞和刺激肾脏的其他条件 纤维发生诱导实质分化状态的变化 成纤维细胞的特征是分泌的明显增加 胶原蛋白类别以及收缩表型相关的获取 用A-Smooth肌肉肌动蛋白的表达：“肌纤维细胞”。许多 完整肾脏的研究暗示了血管紧张素II，TGF-B1和其他 代理是肌纤维细胞分化所需的关键因素。相比之下， 研究成纤维细胞和其他细胞中A平滑肌肌动蛋白表达的研究 文化产生了矛盾的结果。初步证据 此应用表明TGF-BI对 培养中哺乳动物成纤维细胞的表达。这个项目的重点将 确定DNA调节元件和DNA结合蛋白 A-Smooth肌肉肌动蛋白启动子负责尿阻塞依赖性 成纤维细胞中的表达。 BALB/C鼠标成纤维细胞将由稳定标记 绿色荧光蛋白（GFP）的表达，并注入 合成小鼠，将评估它们的阻塞依赖性诱导 A-Smooth肌肉肌动蛋白（SMA）的表达。然后，明显的成纤维细胞将是 使用萤火虫和 Renilla荧光素酶双重报告基因系统以识别DNA元素 对于阻塞依赖性表达必不可少的。移动转移测定法和其他 然后，技术将用于识别DNA结合蛋白相互作用 这些网站。最后，合成纤维细胞系统将用于 在阻塞和 对侧肾脏，以识别细胞外基质的决定因素 积累。这些研究将试图确定因素 尿路阻塞中的肾小管间隙纤维化的启动。这 结果可能会导致确定逮捕新的治疗靶标 并逆转间质纤维化。