FIBROBLAST ACTIVATION IN URINARY OBSTRUCTION

说明(项目摘要) 尿路梗阻和其他刺激肾脏的情况 纤维化诱导实质细胞分化状态的改变 成纤维细胞的特征是分泌明显增加的几种 胶原蛋白的种类，以及与收缩表型相关的获得 表达α-平滑肌肌动蛋白：“肌成纤维细胞”。一批 对完整肾脏的研究表明，血管紧张素II、转化生长因子-β1和其他 药物是肌成纤维细胞分化的关键因素。相比之下， α-平滑肌肌动蛋白在成纤维细胞等细胞中表达的研究 文化产生了相互矛盾的结果。提交的初步证据 这一应用表明，转化生长因子-BI对 哺乳动物成纤维细胞在培养中的表达。该项目的重点将是 鉴定细胞中的DNA调控元件和DNA结合蛋白 α-平滑肌肌动蛋白启动子与尿路梗阻依赖性 在成纤维细胞中表达。BALB/c小鼠成纤维细胞将标记为稳定 绿色荧光蛋白(GFP)的表达，并注射到小鼠肾脏 同基因小鼠，在那里它们将被评估为梗阻依赖诱导 α-平滑肌肌动蛋白(SMA)的表达。标记的成纤维细胞将被 稳定地转染SMA启动子-报告基因的构建 雷尼拉荧光素酶双报告系统用于DNA分子的鉴定 对于梗阻依赖的表达是必不可少的。迁移率变化分析和其他 然后将使用技术来鉴定相互作用的DNA结合蛋白 在这些网站上。最后，同基因成纤维细胞系统将被用于 多组氨酸标记蛋白在梗阻血管间质和血管间质中的表达 对侧肾脏，以确定细胞外基质的决定因素 积累。这些研究将试图找出 尿路梗阻时肾小管间质纤维化的发生。这个 结果可能导致确定新的治疗目标以进行逮捕 逆转间质纤维化。

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.