Shi W K, Yao Z
Shi Yan Sheng Wu Xue Bao. 1989 Jun;22(2):213-23.
Our previous study indicated that polypeptides isolated from acid/ethanol extracts of solid tumors of a cloned F9-3 embryonal carcinoma cells by Bio-Gel P60 column chromatography were found to be able to stimulate anchorage independent growth of either NIH 3T3 cells or NRK 49 F cells in soft agar. The major peak of active elute had a molecular weight of about 15 kDa as determined by SDS-polyacrylamide gel electrophoresis. In the present report we further isolated and purified the active compound corresponding to molecular weight of 15 kDa by gel filteration on Bio-Gel P10 column (Fig. 1) and then by high pressure liquid chromatography (Fig. 2). It was found that the purified 15 kDa molecules showed some properties similar to transforming growth factor-beta (TGF-beta): 1. Colony-stimulating activity in soft agar can be induced in NRK 49 F cells only in the presence of mouse epidermal growth factor (EGF) (Plate I); 2. Increase in relative uptake of 3H-thymidine in NRK 49 F cells occurred in the presence of EGF, but with the same amount of EGF, not much change in 3H-thymidine incorporation could be found with further increasing amounts of purified 15 kDa molecules (Fig. 3); 3. Like human blood platelets derived TGF-beta, inhibition effect on the growth of mink lung epithelial cells (CCL/64) can also be exhibited by purified 15 kDa molecules (Fig. 4). In addition, using ELISA procedure, we have also demonstrated that the 15 kDa molecules had immunological reactivity with the antibody raised against a synthetic oligopeptide identical to the N-terminal residues 1-29 of TGF-beta 1 from human blood platelets (Fig.5). Thus, the 15 kDa molecules isolated from mouse F9-3 embryonal carcinoma cells appeared to share some common antigenic determinants with human TGF-beta 1 molecule. These results taken together provide strong support for the existence of TGF-beta like growth factor in mouse embryonal carcinoma cells.
我们之前的研究表明，通过Bio-Gel P60柱层析从克隆的F9-3胚胎癌细胞实体瘤的酸/乙醇提取物中分离出的多肽，能够在软琼脂中刺激NIH 3T3细胞或NRK 49 F细胞的非锚定依赖性生长。通过SDS-聚丙烯酰胺凝胶电泳测定，活性洗脱液的主峰分子量约为15 kDa。在本报告中，我们通过在Bio-Gel P10柱上进行凝胶过滤（图1），然后通过高压液相色谱（图2），进一步分离并纯化了对应分子量为15 kDa的活性化合物。结果发现，纯化的15 kDa分子表现出一些与转化生长因子-β（TGF-β）相似的特性：1. 仅在存在小鼠表皮生长因子（EGF）的情况下，NRK 49 F细胞在软琼脂中才能诱导集落刺激活性（图版I）；2. 在存在EGF的情况下，NRK 49 F细胞中3H-胸腺嘧啶核苷的相对摄取量增加，但在相同量的EGF存在下，随着纯化的15 kDa分子量的进一步增加，3H-胸腺嘧啶核苷掺入量变化不大（图3）；3. 与源自人血小板的TGF-β一样，纯化的15 kDa分子也能对貂肺上皮细胞（CCL/64）的生长产生抑制作用（图4）。此外，使用ELISA方法，我们还证明了15 kDa分子与针对与人血小板TGF-β1 N端1-29位残基相同的合成寡肽产生的抗体具有免疫反应性（图5）。因此，从小鼠F9-3胚胎癌细胞中分离出的15 kDa分子似乎与人TGF-β1分子具有一些共同的抗原决定簇。综合这些结果，有力地支持了小鼠胚胎癌细胞中存在TGF-β样生长因子。
