Studies on autocrine system for migration factor for smooth muscle cells

用培养的兔和大鼠主动脉平滑肌细胞进行实验，结果表明：1.兔和大鼠主动脉平滑肌细胞的条件培养液具有较高的迁移活性。其活性是纯化的血小板衍生生长因子(PDGF)的数倍。我们将该因子命名为平滑肌细胞衍生生长因子(SDGF)。SDGF具有趋化性，但不具有趋化性。在酸化、碱化、冻融、还原等处理中较稳定，在加热、酸化等处理中不稳定。对于截留孔径为3500的膜，它不能被溶解。这种活性不能被抗PDGF或纤维连接蛋白的抗体中和。这些结果表明，SDMF是一种不同于已知的平滑肌细胞迁移因子的新因子。动脉粥样硬化内膜的血管内膜细胞分泌的SDMF是正常内膜的2-3倍。转化生长因子-β预先孵育可减少血管内皮细胞分泌SDMF。纯化：将SDMF吸附到肝素柱上，用1M氯化钠洗脱。SDMF的色谱峰收集、二次干燥、再干燥，然后进行高效液相色谱(高效液相色谱-Superose 6)。在分子大小为400kD和40kD时出现两个峰。后一峰用含有十二烷基硫酸钠的洗脱液再次进入高效液相色谱-超糖12柱。在分子大小为25KD和小于500D处出现两个峰。这种提纯现在正在进行中。

Following results were obtained using cultured rabbit and rat aortic smooth muscle cells :1. Conditioned media from cultured rabbit and rat aortic smooth muscle cells showed high migration activity for smooth muscle cells. The activity was several times as high as that of purified platelet derived growth factor (PDGF). We named this factor smooth muscle cell derived growth factor (SDGF).2. SDGF was chemotactic but not chemokinetic. It was stable at the treatments such as acidification, alkalization, freezing and thawing, and reduction but was unstable at the treatments such as heating and tripsinization. It was not dializable against the membrane with cutoff pore size 3500. The activity was not neutralized by the antibodies against PDGF or fibronectin. These results suggested that SDMF was a new factor different from known migration factors for smooth muscle cells.3. Secretion of SDMF was 2-3 fold higher in the smooth muscle cells from atherosclerotic intima than that in the smooth muscle cells from normal media.4. Preincubation with transforming growth factor- beta reduced secretion of SDMF from smooth muscle cells.5. Purification : SDMF was adsorbed to heparin-column and eluted by 1M NaC1. The peak of SDMF was collected, dialized, reophilized, and then subjected to a high performance liquid chromatography (HPLC superose 6). Two peaks were obtained at molecular size 400KD and 40KD. The latter peak was again sudjected to HPLC superose 12 with eluate containing SDS. Two peaks were obtained at molecular size 25KD and less than 500D. This purification is now in progress.