BIOLOGIC ASSEMBLY AND DEGRADATION OF FIBRILLAR STRUCTURES OF THE ECM

ECM 纤维结构的生物组装和降解

• 批准号: 6104730
• 负责人: RICHARD MAYNE
• 金额: $ 6.85万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6104730
• 关键词: animal tissue; blocking antibody; collagen; collagenase; elastin; electron microscopy; extracellular matrix; extracellular matrix proteins; fibroblasts; gingiva; high performance liquid chromatography; human tissue; immunocytochemistry; immunofluorescence technique; light microscopy; monoclonal antibody; mucopolysaccharides; protein degradation; protein sequence; protein structure; recombinant proteins; tissue /cell culture; transmission electron microscopy

Experiments will be performed to investigate the structure and degradation of the extracellular matrix assembled by human gingival fibroblasts in cell culture. It will initially be shown that gingival fibroblasts can assemble the three major fibrillar systems found in most connective tissues: namely, the interstitial collagen fibrils assembled from type I, III and V collagens (and possibly type XII and/or type XIV collagen), the elastin (beaded, oxytalan) fibers and the microfibrils of type VI collagen. Monoclonal or polyclonal antibodies will be used to demonstrate the assembly of these fibrils and, where necessary, additional monoclonal antibodies will be prepared (e.g. to human type V collagen). Biochemical analyses will be performed to determine the relative proportions of type I, III and V collagens present in the matrix. Experiments will be performed in which the cells, cell membranes and soluble proteins of the matrix will be extracted by mild detergent (Triton X-100) to leave a fibrillar matrix whose structure will be investigated by i) transmission electron microscopy, ii) electron microscopic immunolocalization using a second antibody coupled to colloidal gold, iii) rotary shadowing of a homogenate of the matrix. We will determine that the structure of the three fibril systems in the extracellular matrix are unaffected by detergent extraction. We will also explore the effects of removal of glycosaminoglycans from the matrix by digestion with chondroitinase ABC or hyaluronidase. Once these experiments are completed, gingival fibroblasts will be grown and the matrix radioactively-labeled with [3H]glycine. The matrix preparation will be cut into small squares and used as a substrate for the three metalloproteinases thought to be required for successful matrix degradation: namely, collagenase, stromelysin and 72 kD gelatinase. We presently have available recombinant collagenase and stromelysin and recombinant 72 kD gelatinase will be obtained from Dr. Jeffrey Engler's laboratory, UAB. The release of peptide fragments from the matrix will be examined initially by fluorography and large-scale preparations will be made of any significant peptides that are released. Initial identification of peptides will be based on N-terminal amino acid sequencing after blotting onto Immobilon membrane. Cultures will be examined by light and electron microscopy and, additionally by rotary shadowing, to determine the effects of each enzyme either separately or in combination on the structure of the three fibril systems. These experiments will explore the enzymatic requirements for the degradation of a matrix which closely resembles the matrix as it exists in the gingiva. Nothing is known of the requirements for the degradation either of the type VI fibrils or the elastin (oxytalan) fibers and yet both are key fibrils of the matrix. The results will provide important new insights into the degradation of naturally occurring fibril systems in a manner which previously has not been achieved.

将进行实验以研究其结构和 人牙龈组装的细胞外基质的降解 细胞培养物中的成纤维细胞。 最初将显示牙龈 成纤维细胞可以组装大多数细胞中发现的三种主要纤维系统 结缔组织：即间质胶原纤维聚集而成 来自 I、III 和 V 型胶原蛋白（可能还有 XII 型和/或 XIV 型） 胶原蛋白）、弹性蛋白（珠状、oxytalan）纤维和微纤维 VI 型胶原蛋白。 单克隆或多克隆抗体将用于 展示这些原纤维的组装，并在必要时， 将制备额外的单克隆抗体（例如针对人 V 型 胶原）。 将进行生化分析以确定 I、III 和 V 型胶原蛋白的相对比例 矩阵。 将进行以下实验：细胞、细胞膜 基质的可溶性蛋白质将被温和的洗涤剂提取 （Triton X-100）留下纤维状基质，其结构为 通过 i) 透射电子显微镜、ii) 电子显微镜进行研究 使用偶联的第二抗体进行显微免疫定位 胶体金，iii) 基质匀浆的旋转阴影。 我们 将决定三个原纤维系统的结构 细胞外基质不受洗涤剂提取的影响。 我们将 还探讨了从基质中去除糖胺聚糖的影响 通过软骨素酶 ABC 或透明质酸酶消化。 一旦这些 实验完成后，牙龈成纤维细胞将会生长， 用[3H]甘氨酸放射性标记的基质。 基质制备 将被切成小方块并用作三个的基板 金属蛋白酶被认为是成功基质所必需的 降解：即胶原酶、溶基质素和72 kD 明胶酶。 我们 目前已有可用的重组胶原酶和溶基质素 重组 72 kD 明胶酶将从 Jeffrey Engler 博士获得 实验室，UAB。 肽片段从基质中的释放将 最初通过荧光照相术进行检查，并进行大规模制备 由释放的任何重要肽组成。 最初的 肽的鉴定将基于 N 端氨基酸 印迹到 Immobilon 膜上后进行测序。 文化将是 通过光学和电子显微镜以及旋转显微镜检查 阴影，以单独或确定每种酶的作用 结合三种原纤维系统的结构。 这些 实验将探索降解的酶促要求 与存在于矩阵中的矩阵非常相似的矩阵 牙龈。 对于降解的要求也一无所知 VI 型原纤维或弹性蛋白（oxytalan）纤维，但两者都是 基质的关键原纤维。 研究结果将提供重要的新 对自然发生的原纤维系统降解的见解 以前从未实现过的方式。