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

调节生肌细胞增殖和分化的体液因子的研究：鸡胚提取物中因子的纯化和表征。

• 批准号: 63540591
• 负责人: KIMURA Ichiro
• 金额: $ 1.34万
• 依托单位: Waseda University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63540591/
• 关键词: growth factors; chick embryo extract; muscle differentiation; myogenesis; myogenic cells; myoblasts; cell proliferation; fibroblast growth factor; 筋形成; ヘパリン結合性; 線維芽細胞成長因子; 細胞分化; 増殖因子

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

从鸡胚提取物（EE）中分离纯化了一种促进成肌细胞增殖的体液因子，经阳离子交换层析和肝素亲和层析，纯化倍数约为10^5倍。该因子用2 M NaCl从肝素-琼脂糖凝胶中洗脱，是一种单链多肽，表观分子量为15，000至17，000。它在亚纳克水平上具有触发增殖的活性，从而暂时延迟成肌细胞的融合。它还在类似的有效浓度范围内刺激鹌鹑成纤维细胞的增殖。对于成肌细胞和成纤维细胞的因子的剂量反应是定量和定性与牛垂体成纤维细胞生长因子。这些观察有力地表明， ...更多信息 经SDS-聚丙烯酰胺凝胶电泳分析，发现该因子有两条主要带。因此，仍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。然后，我们尝试进一步纯化因子的高效液相色谱采用反相柱。另外，我们尝试建立一种更简便、可靠的检测系统来代替我们使用的禽类原代成肌细胞的常规检测系统。我们研究了鹌鹑成肌细胞系NIBB/QM的成肌分化的培养条件，该细胞系具有温度依赖性。提出了严格调整细胞密度等培养条件，NIBB/QM可作为检测细胞的可能性。少

项目成果

Ichiro Kimura: "Occurence of degradation products of chicken transferrin in murine circulating blood." Jap.J.Pharmacol.51. 397-401 (1989)

Ichiro Kimura：“小鼠循环血液中鸡转铁蛋白降解产物的出现。”

Ichiro Kimura: "Further purification of a fibroblast growth factorーlike factor from chick embryo extract by heparinーaffinity chromatography." In Vitro. 25. 236-242 (1989)

Ichiro Kimura：“通过肝素亲和层析从鸡胚提取物中进一步纯化成纤维细胞生长因子样因子。”25. 236-242 (1989)

Ichiro Kimura: "Further purification of a fibroblast growth factor-like factor from chick embryo extract by heparin-affinity chromatography." In Vitro. 25. 236-242 (1989)

Ichiro Kimura：“通过肝素亲和层析从鸡胚提取物中进一步纯化成纤维细胞生长因子样因子。”

Ichiro Kimura,Yae Goto,Eijiro Ozawa: "Further purification of a fibroblast growth factor-like factor from chick enbryoextract by heparin-affinity chromatography." In Vitro Cell.Develop.Biol.25. 236-242 (1989)

Ichiro Kimura、Yae Goto、Eijiro Ozawa：“通过肝素亲和层析从鸡胚提取物中进一步纯化成纤维细胞生长因子样因子。”

木村 一郎: "トランスフェリン" 臨床検査. 34. 1549-1551 (1990)

木村一郎：“转铁蛋白”临床测试 34。1549-1551 (1990)。