Martin J L, Baxter R C
Department of Medicine, University of Sydney, New South Wales, Australia.
Endocrinology. 1988 Oct;123(4):1907-15. doi: 10.1210/endo-123-4-1907.
We have characterized the insulin-like growth factor-binding protein (IGF-BP) produced by neonatal human skin fibroblasts in monolayer culture using antibodies specific for the acid-stable subunit of the 150K GH-dependent IGF-BP complex, BP-53, and the amniotic fluid IGF-BP, BP-28. Fibroblasts produced 65.3 +/- 10.4 ng/ml.72 h (SE; n = 6) immunoreactive BP-53 in serum-free medium; this was stimulated by increasing fetal bovine serum in the medium up to 385.3 +/- 49.0 ng/ml.72 h at 10% serum. Epidermal growth factor (EGF) also caused dose-dependent stimulation of BP-53 production, with a maximal effect (3-fold increase) at 30 ng/ml EGF. No immunoreactive BP-28 production was detectable in the presence or absence of serum or EGF. Neutral gel chromatography of serum-free medium revealed a peak of immunoreactive BP-53 at about 50K, with a smaller species at 20-30 K. Serum- and EGF-stimulated cells produced higher levels of about 50K BP-53, and an additional peak of immunoreactivity at 150K was present in serum-stimulated, but not EGF-stimulated, samples. Comparison of IGF-I and IGF-II binding by fibroblast BP-53 revealed slightly higher IGF-II than IGF-I binding, and association constants of 3-4 x 10(10) liter/mol for both IGFs, similar to BP-53 from human plasma. Affinity labeling of acid-stripped medium followed by nonreduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed specifically cross-linked IGF-binding species of 60K (identical to labeled plasma BP-53), 42K, and 37K. Only the 60K and 42K complexes were precipitable by antiserum to plasma BP-53, and none was precipitable by anti-BP-28 serum, suggesting that the 37K band might represent a third class of IGF-BP. We conclude that neonatal skin fibroblasts produce no BP-28, but do produce two IGF-BPs immunologically homologous to human plasma BP-53, one of which shows size and IGF-binding characteristics identical to the plasma protein.
我们利用针对150K生长激素依赖性胰岛素样生长因子结合蛋白（IGF-BP）复合物的酸稳定亚基BP-53以及羊水IGF-BP（BP-28）的特异性抗体，对单层培养的新生儿人皮肤成纤维细胞产生的IGF-BP进行了特性分析。成纤维细胞在无血清培养基中72小时产生65.3±10.4 ng/ml（标准误；n = 6）的免疫反应性BP-53；在培养基中添加胎牛血清，最高可达10%血清时，其产量可增至385.3±49.0 ng/ml.72小时。表皮生长因子（EGF）也能引起BP-53产量的剂量依赖性刺激，在30 ng/ml EGF时达到最大效应（增加3倍）。无论有无血清或EGF，均未检测到免疫反应性BP-28的产生。对无血清培养基进行中性凝胶色谱分析，结果显示免疫反应性BP-53在约50K处出现一个峰值，在20 - 30K处有一个较小的峰。血清和EGF刺激的细胞产生的约50K BP-53水平更高，在血清刺激而非EGF刺激的样品中，150K处还出现了一个额外的免疫反应峰。对成纤维细胞BP-53与IGF-I和IGF-II结合的比较显示，其与IGF-II的结合略高于与IGF-I的结合，两种IGF的结合常数均为3 - 4×10(10)升/摩尔，与人血浆中的BP-53相似。对酸处理后的培养基进行亲和标记，然后进行非还原十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳，结果显示特异性交联的IGF结合条带分别为60K（与标记的血浆BP-53相同）、42K和37K。只有60K和42K复合物可被抗血浆BP-53抗血清沉淀，而抗BP-28血清均不能沉淀，这表明37K条带可能代表第三类IGF-BP。我们得出结论，新生儿皮肤成纤维细胞不产生BP-28，但确实产生两种与人类血浆BP-53免疫同源的IGF-BP，其中一种在大小和IGF结合特性上与血浆蛋白相同。
