ISOLATION OF THE SERUM FACTOR RESPONSIBLE FOR PSEUDOXANTHOMA ELASTICUM

分离导致弹性假黄瘤的血清因子

• 批准号: 7959637
• 负责人: OLIVIER LE SAUX
• 金额: $ 1.49万
• 依托单位: UNIVERSITY OF HAWAII AT MANOA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7959637
• 关键词: Animal Model; Aorta; Biological Assay; Cardiovascular system; Cell Line; Centers of Research Excellence; Characteristics; Computer Retrieval of Information on Scientific Projects Database; Culture Media; Defect; Deposition; Disease model; Elastic Fiber; Elastin; Electron Microscopy; Event; FBN1; Fiber; Fibroblasts; Fractionation; Funding; Goals; Grant; Homeostasis; Human; In Vitro; Individual; Institution; LOX gene; Mass Spectrum Analysis; Molecular; Nature; Patients; Peptide Hydrolases; Production; Proteins; Protocols documentation; Pseudoxanthoma Elasticum; Research; Research Personnel; Resources; Serum; Skin; Smooth Muscle Myocytes; Source; Staging; Testing; United States National Institutes of Health; base; calcification; fibulin-4; in vivo; structural biology

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Objectives. The long-term goal of this study is to better understand the remarkable biology and structural organization of elastic fibers. This general goal is best achieved by studying these fibers in vitro and in vivo using both human and/or animal models. The specific goal of our project is to identify and characterize the molecular events leading to elastic fiber alterations and calcification in pseudoxanthoma elasticum (PXE) as a pertinent disease model that should help elucidate the dynamics governing the homeostasis of elastic fiber proteins. Specific Aim 1: Determine the defect in the assembly of elastic fiber caused by PXE serum in vitro. We will use primary cell lines of skin fibroblasts and normal aorta smooth muscle cells with culture medium supplemented with serum from PXE patients or normal individuals. The principal elastic fiber components (elastin, fibulin-4, -5, fibrillin-1, -2, MAGP1, LOX and LOXL) will be examined by immunohistology and quantified during various stages of elastic fiber deposition, from pre- to post-confluence. Structural alterations will be evaluated by electron microscopy. The production of elastic fiber-specific proteases by fibroblasts and smooth muscle cells in the presence of PXE serum will also be evaluated by zymography. Specific Aim 2: Isolate serum factor(s) interfering with elastic fiber formation. The nature of the PXE serum factor(s) is unknown. We propose to determine the molecular characteristics of the PXE serum active component(s) and establish an isolation protocol based on chromatographic fractionations followed by mass spectroscopy analysis. The fractions will be tested using in vitro functional assays.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 目标。这项研究的长期目标是更好地了解弹性纤维的显着生物学和结构组织。通过使用人和/或动物模型在体外和体内研究这些纤维，可以最好地实现这一总体目标。我们项目的具体目标是识别和表征分子事件，导致弹性纤维弹性的弹性改变和钙化弹性（PXE）为一种相关疾病模型，该模型应有助于阐明控制弹性纤维蛋白稳态的动力学。 特定目标1：确定由PXE血清在体外引起的弹性纤维组装中的缺陷。 我们将使用皮肤成纤维细胞和正常主动脉平滑肌细胞的原代细胞系，并配有培养基，并补充了来自PXE患者或正常个体的血清。主弹性纤维成分（弹性蛋白，fibulin-4，-5，Fibrillin-1，-2，-2，Magp1，Lox和LoxL）将通过免疫组织学检查，并在弹性纤维沉积的各个阶段进行量化，从弹性纤维沉积的各个阶段，从前到综合后到弹性。结构改变将通过电子显微镜评估。在PXE血清存在下，成纤维细胞和平滑肌细胞的弹性纤维特异性蛋白酶的产生也将通过Zymography评估。 特定的目标2：分离的血清因子干扰弹性纤维形成。 PXE血清因子的性质未知。我们建议确定PXE血清活性成分的分子特性，并基于色谱分馏，然后进行质谱分析，建立隔离方案。将使用体外功能测定法对级分进行测试。