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

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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