Collagen synthesis by bovine aortic endothelial cells in culture.
Collagen synthesis by bovine aortic endothelial cells in culture.
复制标题
培养物中牛主动脉内皮细胞的胶原合成。
作者：
H. Sage;E. Crouch;P. Bornstein
从牛主动脉分离的内皮细胞在培养中合成和分泌III型前胶原。前胶原，这代表了主要的胶原蛋白在培养基中，特异性沉淀的抗体牛III型前胶原，并通过二乙基-氨基乙基纤维素层析纯化。通过与通过胃蛋白酶消化牛皮肤分离的III型胶原直接比较，利用脊椎动物胶原酶、CNBr和肥大细胞蛋白酶产生的肽裂解模式，对胃蛋白酶处理的胶原进行明确鉴定。III型胶原被高度羟基化，具有1.5：1.0的羟脯氨酸/脯氨酸比。脉冲追踪研究表明，前胶原没有被加工成前胶原中间体或胶原。胃蛋白酶处理细胞层，然后在酸性和中性pH下进行盐分级分离，产生了几种对细菌胶原酶敏感的组分，这些组分在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上与通过类似技术从人胎盘纯化的α A、α B和IV型胶原链共迁移。牛主动脉内皮细胞还分泌纤连蛋白和一种细菌胶原酶不敏感糖蛋白，其还原后在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（使用前胶原分子量标准品）上的分子量为135，000，并且不能被冷不溶性球蛋白或α 2-巨球蛋白的抗体沉淀。这些细胞的胶原蛋白生物合成为研究蛋白质分泌的极性和细胞与细胞外基质的附着提供了一个有趣的模型系统。III型胶原在内皮下的存在和这种蛋白质与纤连蛋白和血小板的特异性相互作用表明，这种胶原参与内皮细胞损伤后血栓形成。
Endothelial cells isolated from bovine aorta synthesize and secrete type III procollagen in culture. The procollagen, which represents the major collagenous protein in culture medium, was specifically precipitated by antibodies to bovine type III procollagen and was purified by diethyl-aminoethylcellulose chromatography. Unequivocal identification of the pepsin-treated collagen was made by direct comparison with type III collagen isolated by pepsin digestion of bovine skin, utilizing peptide cleavage patterns generated by vertebrate collagenase, CNBr, and mast cell protease. The type III collagen was hydroxylated to a high degree, having a hydroxyproline/proline ratio of 1.5:1.0. Pulse-chase studies indicated that the procollagen was not processed to procollagen intermediates or to collagen. Pepsin treatment of cell layers, followed by salt fractionation at acidic and neutral pH, produced several components which were sensitive to bacterial collagenase and which comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with alpha A, alpha B, and type IV collagen chains purified from human placenta by similar techniques. Bovine aortic endothelial cells also secreted fibronectin and a bacterial collagenase-insensitive glycoprotein which, after reduction, had a molecular weight of 135,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (using procollagen molecular weight standards) and which was not precipitable by antibodies to cold-insoluble globulin or to alpha 2-macroglobulin. Collagen biosynthesis by these cells provides an interesting model system for studying the polarity of protein secretion and the attachment of cells to an extracellular matrix. The presence of type III collagen in the subendothelium and the specific interaction of this protein with fibronectin and platelets suggest the involvement of this collagen in thrombus formation following endothelial cell injury.