Speir E, Sasse J, Shrivastav S, Casscells W
Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Cell Physiol. 1991 May;147(2):362-73. doi: 10.1002/jcp.1041470223.
The activity of acidic and basic fibroblast growth factor-like mitogens (aFGF, bFGF) extracted from cultured bovine aortic endothelial (BAEC) and rat aortic smooth muscle cells (SMC) was compared with that of freshly isolated cells from the same tissues. Extracts of subendothelial extracellular matrix (ECM) and cell lysates of cultured BAEC contained 4-fold more bFGF-like activity than the extracts of fresh cells. ECM and cell lysates of SMC yielded 10-fold more bFGF-like activity than the fresh cell lysates. We consistently find aFGF-like activity in both cell types. In the case of BAEC, cultured cells and ECM contained 3-fold more aFGF-like activity when compared with freshly isolated cells, whereas in cultured SMC, aFGF-like activity in cell and ECM extracts was 8-fold higher than in fresh cell extracts. The mitogens extracted from cell lysates and from the ECM are closely related to aFGF or bFGF by the criteria that they bind to heparin-sepharose and elute at 1.1 M (aFGF) or 1.5 M (bFGF) NaCl, have molecular weights of about 18,000, and react with anti-aFGF (1.1 M), or anti-bFGF (1.5 M) antibodies when analyzed by Western blots and by radioimmunoassay specific for aFGF and bFGF. This mitogenic activity is inhibited by neutralizing antibodies to aFGF and bFGF. In addition, the column fractions are potent mitogens for Balb/c 3T3 fibroblasts. Acidic and basic FGF-like mitogenic activity could also be extracted from the cell nuclei. The subcellular localization of both FGFs was visualized in both nuclei and cytoplasm with immunoperoxidase. Compared with primary SMC, secondary SMC had an increased capacity to bind 125IaFGF to high affinity receptors, while binding to freshly isolated BAEC and SMC was negligible. We conclude that FGFs are present at low levels in freshly isolated cells and that propagation in cell culture provides a stimulus for production of these mitogens.
将从培养的牛主动脉内皮细胞（BAEC）和大鼠主动脉平滑肌细胞（SMC）中提取的酸性和碱性成纤维细胞生长因子样促细胞分裂剂（aFGF、bFGF）的活性，与从相同组织中新鲜分离的细胞的活性进行了比较。内皮下细胞外基质（ECM）提取物和培养的BAEC的细胞裂解物中bFGF样活性比新鲜细胞提取物高4倍。SMC的ECM和细胞裂解物产生活性比新鲜细胞裂解物高10倍的bFGF样活性。我们在两种细胞类型中均持续检测到aFGF样活性。就BAEC而言，与新鲜分离的细胞相比，培养细胞和ECM中aFGF样活性高3倍，而在培养的SMC中，细胞和ECM提取物中的aFGF样活性比新鲜细胞提取物高8倍。从细胞裂解物和ECM中提取的促细胞分裂剂与aFGF或bFGF密切相关，依据如下：它们与肝素琼脂糖结合，并在1.1M（aFGF）或1.5M（bFGF）NaCl浓度下洗脱，分子量约为18,000，并且在通过蛋白质免疫印迹法和针对aFGF和bFGF 的放射免疫测定法分析时，与抗aFGF（1.1M）或抗bFGF（1.5M）抗体发生反应。这种促有丝分裂活性被针对aFGF和bFGF的中和抗体所抑制。此外，柱层析级分对Balb/c 3T3成纤维细胞是有效的促细胞分裂剂。酸性和碱性FGF样促有丝分裂活性也可从细胞核中提取。两种FGF在细胞核和细胞质中的亚细胞定位通过免疫过氧化物酶法可见。与原代SMC相比，传代SMC与125I-aFGF结合到高亲和力受体的能力增强，而与新鲜分离的BAEC和SMC的结合可忽略不计。我们得出结论，FGF在新鲜分离的细胞中含量较低，并且细胞培养中的传代对这些促细胞分裂剂的产生起到刺激作用。
