CONTROL OF COLLAGENASE IN HUMAN FIBROBLAST CULTURES

胶原酶在启动间质降解中起关键作用 胶原蛋白存在于各种形态发生的重要事件中。我们将使用 培养的皮肤成纤维细胞检测胶原酶的刺激作用 蛋白质，包括20 kDa的表皮细胞因子和血小板衍生的生长 因子对这些靶细胞表达胶原酶的影响。我们会 评估对这些多肽激动剂的反应是否是发育的 通过检测(I)酶所反映的胶原酶表达来调节 免疫活性酶蛋白的活性、合成及其在细胞内的变化 可杂交的mRNA和(II)靶成纤维细胞的有丝分裂反应 与细胞因子有关。我们将使用培养的不同阶段的成纤维细胞 发展包括从遗传上的“衰老综合征”来解决是否 这两种反应反映了不同的机械途径。这些 细胞因子影响血管内皮细胞生长的机制研究 胶原酶的表达将通过确定基因产物是否 胶原酶本身，可以通过探测以任何方式影响自己的合成 其他激动剂对正常细胞和不同类型细胞的作用 大疱性表皮松解症，并通过检测一种核酸的作用 维甲酸和糖皮质激素等抑制 胶原酶合成。 因为有证据表明胶原酶的结构突变形式 隐性营养不良性大疱性表皮松解症(RDEB)，我们将采用培养 本病成纤维细胞作为胶原酶mRNA的来源以克隆 疑似突变的胶原酶基因。作为隔离和隔离的探针 RDEB基因克隆的初步鉴定我们将采用 正常人皮肤胶原酶pCOL 185.2全长基因克隆。我们的 目标是集中精力定义导致突变的部位(S) 结构改变的蛋白质，这样我们就可以更好地理解 调节机制，提供改进的诊断和遗传咨询， 并在这场毁灭性的灾难中设计合理的治疗方式 遗传性皮肤病。

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.