Isolation and Identification of Myotube-forming Stimulating Factor on Skeletal Muscle of Domestic Animals

从小鼠、大鼠、鸡和猪的骨骼肌中分离的原代肌源性细胞的大量体外研究表明，细胞的生长和终末分化受多种生长因子的控制。然而，牛成肌细胞的成肌作用，即从细胞增殖到终末分化的转变，并不总是被理解。本研究旨在分离和鉴定一种肌管形成刺激因子（MTF），并探讨其在牛成肌细胞成肌过程中的作用。本实验主要进行了以下几个方面的研究：（1）牛成肌细胞体外成肌作用的建立。采用199培养基、Dulbecco改良Eagle培养基和FCS按8：2：1混合，并添加1.5%猪胎儿提取物（SFE）的培养基建立牛成肌细胞培养体系。采用差速贴壁法和细胞松弛素B tre筛选相结合的方法富集牛成肌细胞 关于我们 补偿SFE、胰岛素（10 μ <-6>M）和IGF-1（10 μ <-7>M-10 μ <-8>M）通过成肌细胞融合引起肌管的显著增加。(2)MTF的分离和鉴定：在胎儿肌肉、肺和肾的SFE中检测到MTF活性。通过凝胶过滤，在3- 10 kD和10- 30 kD组分中检测到活性。通过肝素亲和层析，用0.9-1.2M NaCl溶液洗脱MTF，并鉴定为不同于IGF-1的单一蛋白带。(3)MTF对牛成肌细胞AchE和AchR表达的影响AchE和AchR在定向分化的牛成肌细胞上均有表达，并在G_0期分化。MTF和IGF-1可诱导牛成肌细胞AchE和AchR的表达。(4)克隆牛成肌细胞的建立。在牛成肌细胞殖民地培养中建立了克隆牛成肌细胞Bmb-1和Bmb-2。用抗结蛋白抗体染色，形成肌管。用MTF和IGF-1刺激Bmb-2克隆细胞的肌管形成。(5)原代培养牛成肌细胞三维组织的形成。将在琼脂平板上的成肌细胞培养物中聚集成松散结合的团块的细胞聚集体转移到Culstir烧瓶中，并以约70 rpm的转速培养。肿块的致密性逐渐增加，形成大小不等（直径250-500 μ m）的多细胞球体。通过MTF和IGF-1加速球状体形成。结果表明，牛成肌细胞在体外培养体系中可以重建三维结构。少

A large number of in vitro studies using primary myogenic cells isolated from the skeletal muscle of mouse, rat, chicken and swine have showed that the cell growth and terminal differentiation are controlled by several growrth factors. However, the myogenesis of bovine myoblasts, the transformation from cell proliferation to terminal differentiation, is not always understood. Present study was conducted to isolate and identify a myotube-forming stimulating factor (MTF) in regulating the myogenesis of bovine myoblasts in vitro and to characterize the possible role. Experiments were conducted as fouows ;(1) Establishment of myogenesis by bovine myoblasts in cultures. Bovine myoblast culture was established by the medium which mixed medium 199, Dulbecco's modified Eagle's medium and FCS in 8 : 2 : 1, and supplemented 1.5% swine fetal extract (SFE). Bovine myoblasts were enriched by the combination procedure of differential adhesion method and a selection of myoblasts by cytochalasin B tre … More atment. SFE,Insulin (10^<-6>M) and IGF-1 (10^<-7> -10^<-8>M) caused a significant increase of myotubes by the myoblast fusion.(2) Isolation and identification of MTF.MTF activity was recognized in SFE of fetal muscle, lung and kidney. By gel filtration, the activity was detected in 3-10kD and 10-30kD fraction. MTF was eluted with 0.9-1.2M NaCI solution by heparin-affinity chromatography and was identified as a single protein band which was different from IGF-1.(3) Effects of MTF on AchE and AchR expression of bovine myoblasts. AchE and AchR were expressed on the committed bovine myoblasts which differentiated in G_0 phase. MTF and IGF-1 induced the AchE and AchR expression of bovine myoblasts.(4) Establishment of cloned bovine myoblasts. Cloned bovine myoblasts, Bmb-1 and Bmb-2, were established in the colony cultures of bovine myoblast. They were stained by anti-desmin antibody and formed myotubes. The myotube formation of Bmb-2 cloned cells was stimulated by MTF and IGF-1.(5) Formation of three-dimensional tissues from primary cultured bovine myoblasts. Cell aggregates which bunched into loosely bound masses in the myoblast cultures on the agar plates were transferred to Culstir flask and cultured at rotation speeds around 70 rpm. The mass increased progressively in compactness and formed various size (250-500 mu m in diameter) of multicellular spheroids. The spheroid formation was accelerated by MTF and IGF-1. The results demonstrated that three-dimensional structures by bovine myoblasts call be reconstituted in culture system. Less