CONTROL OF COLLAGENASE IN HUMAN FIBROBLAST CULTURES

人成纤维细胞培养物中胶原酶的控制

• 批准号: 3481480
• 负责人: EUGENE A BAUER
• 金额: $ 30.57万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-06-01 至 1991-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3481480
• 关键词: collagenase; cyclic AMP; cytogenetics; enzyme induction /repression; enzyme inhibitors; epidermolysis bullosa; fibroblasts; gene expression; genetic regulation; genetic translation; human tissue; laboratory mouse; laboratory rabbit; messenger RNA; orphan disease /drug; secretion; skin disorder; steroid hormone; tissue /cell culture; tritium

Collagenase is critical in initiating the degradation of interstitial collagens in a variety of morphogenetically important events. We shall use cultured skin fibroblasts to examine the effects of collagenase-stimulatory proteins, including a 20 kDa epidermal cytokine and platelet-derived growth factor, on the expression of collagenase by these target cells. We shall assess whether the response to these peptide agonists is developmentally modulated by examining (i) collagenase expression as reflected by enzyme activity, synthesis of immunoreactive enzyme protein and alterations in hybridizable mRNA and (ii) the mitogenic response of the target fibroblasts to the cytokines. We shall use cultured fibroblasts from various stages of development including from the genetic "aging syndromes" to address whether these two responses are reflective of separate mechanistic pathways. These investigations of the mechanisms for the effects of cytokines on collagenase expression will be complemented by defining if the gene product itself, collagenase, can in any way affect its own synthesis, by probing the effects of other agonists on normal cells and cells from various types of epidermolysis bullosa, and by examining the effects at a nucleic acid level of agents such as retinoids and glucocorticoids that inhibit collagenase synthesis. Since there is evidence for a structurally mutant form of collagenase in recessive dystrophic epidermolysis bullosa (RDEB), we shall employ cultured fibroblasts from this disease as a source for collagenase mRNA to clone the putatively mutant collagenase gene. As a probe for the isolation and initial characterization of the RDEB cDNa clones we shall employ a full-length cDNA clone for normal human skin collagenase pCol 185.2. Our goal is to focus intensely on defining the site(s) of mutation leading to the structurally altered protein, so that we can better understand regulatory mechanisms, provide improved diagnosis and genetic counseling, and devise rational therapeutic modalities in this devastating genodermatosis.

胶原酶对于启动间质的降解至关重要 胶原蛋白参与多种形态发生的重要事件。 我们将使用 培养皮肤成纤维细胞以检查胶原酶刺激的效果 蛋白质，包括 20 kDa 表皮细胞因子和血小板衍生生长 影响这些靶细胞表达胶原酶的因素。 我们将 评估对这些肽激动剂的反应是否是发育性的 通过检查（i）酶反映的胶原酶表达来调节 免疫反应酶蛋白的活性、合成和改变 可杂交的 mRNA 和 (ii) 靶成纤维细胞的有丝分裂反应 到细胞因子。 我们将使用不同阶段的培养成纤维细胞 发展包括从遗传“衰老综合症”中解决是否 这两种反应反映了不同的机制途径。 这些 细胞因子影响机制的研究 胶原酶表达将通过定义基因产物是否得到补充 胶原酶本身可以通过探测以任何方式影响其自身的合成 其他激动剂对正常细胞和各种类型细胞的影响 大疱性表皮松解症，并通过检查核酸的影响 类维生素A和糖皮质激素等抑制药物的水平 胶原酶合成。 由于有证据表明胶原酶存在结构突变形式 隐性营养不良性大疱性表皮松解症（RDEB），我们将采用培养 来自这种疾病的成纤维细胞作为胶原酶 mRNA 的来源来克隆 推测突变的胶原酶基因。 作为隔离和探针 RDEB cDNA 克隆的初步表征，我们将采用 正常人皮肤胶原酶 pCol 185.2 的全长 cDNA 克隆。 我们的 目标是集中精力定义导致突变的位点 结构改变的蛋白质，以便我们更好地理解 监管机制，提供改进的诊断和遗传咨询， 并在这场毁灭性的灾难中制定合理的治疗方式 遗传性皮肤病。