PURIFICATION & STUDY OF HUMAN KERATINOCYTE COLLAGENASE

脊椎动物胶原酶，一种中性金属内蛋白酶，裂解 将天然间质胶原化成特定的四分之三和一分之一- 1/4长度的片段，然后容易进一步 蛋白质分解。在皮肤中，胶原酶的过度产生是 认为这与遗传性疾病的发病机制有关 起泡障碍，营养不良性大疱性表皮松解症(RDEB)。 真皮成纤维细胞一直被认为是原发的 人类皮肤中胶原酶的来源，这种酶一直是 经过广泛研究。我们最近记录了 培养的人角质形成细胞产生胶原酶。 角质形成细胞胶原酶在功能上出现 在结构上类似于成纤维细胞酶，尽管 对生产的调整可能会有所不同。这样做的目的是 提案的目的是提纯、进一步描述和评估 正常和RDEB中胶原酶产生的调控 角质形成细胞。胶原酶将从有条件的 佛波醇刺激角质形成细胞的培养液和 以等电点、钙为特征的 要求、热稳定性和部分氨基酸序列 数据。人类皮肤胶原酶抗体将在 兔和建立了一种酶联免疫吸附试验。底物专一性 纯化的酶将用IV型和III型进行评估 胶原蛋白。胶原酶生产的调控将被研究 已知的成纤维细胞胶原酶的刺激物和抑制物， 蛋白激酶C途径类似物和生长因子。这个 细胞-细胞和细胞-基质相互作用的影响 细胞迁移和损伤对胶原酶合成的影响 角质形成细胞将被评估。生产与调制 将研究RDEB角质形成细胞对胶原酶的影响 创伤后胶原酶的免疫细胞化学定位 评估了人体皮肤和皮肤当量模型。

Vertebrate collagenase, a neutral metalloendoproteinase, cleaves native interstitial collagen into specific three-quarter and one- quarter length fragments which are then susceptible to further proteolysis. In the skin, excessive production of collagenase is thought to contribute to the pathogenesis of the inherited blistering disorder, dystrophic epidermolysis bullosa (RDEB). The dermal fibroblast has been considered to be the primary source of collagenase in human skin and the enzyme has been extensively studied. We have recently documented the production of collagenase by human keratinocytes in culture. The keratinocyte collagenase appears functionally and structurally similar to the fibroblast enzyme, although modulation of production may be dissimilar. The goal of this proposal is to purify, further characterize, and evaluate the modulation of collagenase production in normal and RDEB keratinocytes. Collagenase will be purified from conditioned medium from phorbol-stimulated keratinocytes and characterized in regards to the isoelectric point, calcium requirements, thermal stability and partial amino acid sequence data. Antibodies to human skin collagenase will be produced in rabbits and an ELISA developed. Substrate specificity of the purified enzyme will be evaluated with type IV and type VII collagen. Modulation of collagenase production will be studied with known stimulators and inhibitors of fibroblast collagenase, protein kinase C pathway analogues and growth factors. The effect of cell-cell and cell-matrix interactions, cellular migration and cellular injury on synthesis of collagenase by keratinocytes will be assessed. The production and modulation of collagenase by RDEB keratinocytes will be studied and immunocytochemical localization of collagenase in wounded human skin and skin equivalent models evaluated.