Elucidation of the mechanism of caediovascular remodeling and development of therapy by proteomic analysis

本研究的目的是阐明心血管重塑的机制及其治疗进展。通过引入蛋白质组学的研究，主要针对我们过去发现的在心血管重塑中起重要作用的转录因子KLF5，我们对影响心血管疾病发病过程的因子的辅助因子进行了全面的分离，并从功能缺失的角度进行了功能分析。本研究以KLF5为模型，阐明了以KLF5为模型的核内转录因子网络的转录调控机制，旨在开发防治器官重塑的药物。PDGF-A促进平滑肌细胞迁移/增加，是一种在动脉粥样硬化发病中起重要作用的生长因子。以往的研究发现，KLF5是KLF5的靶基因，-71~-55bp的结构域在心血管重构中起重要作用。首先，作为本研究的一个具体方案，我们确定了PDGF-A基因-71~-55bp区域的结合蛋白。我基本上遵循KLF5结合蛋白的筛选。具体地说，我们构建了PDGF-A启动子的KLF5结合部位-71~-55bp的双链DNA，并将其一端生物素基化。我们利用链霉亲和素和生物素的强键将其固定在金属珠的表面。我把细胞的核抽提物放在这里反应，然后往下拉。然后，我们进行了SDS-PAGE，并用凝胶内胰酶消化的方法鉴定了一条特异性条带。研究发现了一些有趣的因素。根据KLF5结合因子的生化特性，从动物水平到细胞水平探讨其致病机制。综上所述，心血管组织对氧化应激防御机制的新机制变得清晰。

The objective of this study is to elucidate the mechanism of cardiovascular remodeling and the development of its therapy. By introducing research proteomics, and, mainly on transcription factor KLF5 which had an important role for the cardiovascular remodeling which we identified in the past, we perform a comprehensive isolation of the co-factors of the factor which affect the pathogenic process of the cardiovascular diseases, a functional analysis by the loss of function system. This study finally elucidates transcription control mechanism by the transcription factor network in the nucleus which assumed KLF5 a model and aims for developing the prevention of the organ remodeling and a cure. PDGF-A promotes a migration/the increase of the smooth muscle cell, and it is a growth factor playing an important role for the onset of the atherosclerosis. It was found to be a target gene of KLF5 by the examination of our past, and it became clear that -71 to -55bp domains were important in cardiovascular remodeling. At first, as a concrete plan in this study, we identify protein which bind -71 to -55bp domains of PDGF-A gene. I follow screening of the KLF5 binding protein basically. To be concrete, we composed the double-stranded DNA of -71 to -55bp domains that were the KLF5 binding site of the PDGF-A promoter and biotinylated the one end. Using streptoavidin and a strong bond of the biotin, we fixed it on the metal beads surface. I reacted the nuclear extract of the cell in this, and pull down. Then, we developed SDS-PAGE and identified a specific band by in-gel trypsin digestion method. Some interesting factors were identified. Based on the biochemical characteristic of the KLF5 binding factor, I pursued the pathogenic mechanism from in the animal level to the cell level. Above all, new mechanism in the defense mechanism of the cardiovascular tissues from oxidation stress became clear.