Study on Humoral Factors which Regulate Proliferation and Differentiation of Myogenic Cells : Purification and Characterization of the Factors from Chick Embryo Extract.

我们试图从鸡胚提取物(EE)中提纯与调节骨骼肌分化有关的体液因子，EE具有很强的肌肉营养作用，经常被用作促进体外成肌细胞生长的介质添加剂。通过阳离子交换层析和肝素亲和层析，一个促进禽成肌细胞增殖的有丝分裂因子已经从EE中纯化了约105倍。该因子由肝素-琼脂糖用2M氯化钠洗脱，为单链多肽，表观分子量在15,000-17,000之间。它在亚纳克水平上是活跃的，可以触发成肌细胞的增殖，从而暂时推迟成肌细胞的融合。在相似的有效浓度范围内，还能刺激成纤维细胞增殖。对于成肌细胞和成纤维细胞，该因子的剂量反应在数量和质量上都与牛垂体成纤维细胞生长因子相当。这些观察结果强烈建议…进一步推测该因子很可能与鸡成纤维细胞生长因子或一个密切相关的分子(S)相对应，并可能参与肌肉发生的调控。因此，目前还不确定该因子是否被其他蛋白质污染，或者是否有两种成分。在此基础上，尝试采用反相柱高效液相色谱对该因子进行进一步纯化。然而，由于因子的失活，我们还没有成功。此外，我们试图建立一个更容易和更可靠的检测体系，以取代我们的常规体系，其中使用的是禽类原代成肌细胞。我们研究了一种具有温度依赖性分化的成肌细胞系NIBB/QM的培养条件。如果严格调整培养条件，如细胞密度等，NiBB/QM有可能被用作检测细胞。较少

We tried to purify humoral factors involved in the regulation of skeletal muscle differentiation from chick embryo extract (EE) which is strongly myotrophic and has often been employed as a medium supplement to promote in vitro myogenesis.A mitogenic factor which promotes avian myoblast proliferation has been purified some 10^5-fold from EE by a combination of cation-exchange chromatography and heparin-affinity chromatography. The factor is eluted from heparin-Sepharose with 2M NaCl and is a single-chain polypeptide with an apparent molecular weight of 15,000 to 17,000. It is active at subnanogram level in triggering the proliferation and thereby delaying temporarily fusion of myoblasts. It also stimulates the proliferation of quail fibroblasts in a similar effective concentration range. For both myoblasts and fibroblasts the dose-response to the factor is quantitatively and qualitatively comparable with that of bovine pituitary fibroblast growth factor. These observations strongly sug … More gest that the factor very probably corresponds to chicken fibroblast growth factor or to a closely related molecule (s) and that it is possibly involved in the regulation of myogenesis.When our purified factor is analyzed on SDS-polyacrylamide gel electrophoresis, two main bands were seen. Therefore, it remains uncertain whether the factor was contaminated with other proteins or whether two components. Then, we tried to further purify the factor by high performance liquid chromatography employing reverse-phase column. However, we have not succeeded as yet because of inactivation of the factor.In addition, we tried to establish a more easy and reliable assay system in place of our routine system in which avian primary myoblasts are used. We examined culture conditions for myogenesis of a quail myogenic cell line NIBB/QM which differentiates temperature-dependently. A possibility was suggested that NIBB/QM could be employed as assay cells if culture conditions such as cell density etc are strictly adjusted. Less