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

利用小鼠、大鼠、鸡和猪骨骼肌分离的原代成肌细胞进行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，牛成肌细胞的肌发生，即从细胞增殖到终末分化的转变，并不总是被理解。本研究分离并鉴定了一种肌管形成刺激因子（MTF）在体外调节牛成肌细胞的肌发生中，并对其可能的作用进行了表征。实验进行如下：(1)牛成肌细胞在培养中的肌生成。牛成肌细胞培养的培养基为199培养基、Dulbecco改良Eagle培养基和FCS按8:2:1的比例混合，并添加1.5%的猪胎儿提取物（SFE）。采用差异黏附法和细胞松弛素筛选法对牛成肌细胞进行了富集。SFE、胰岛素（10^<-6>M）和IGF-1 （10^<-7> -10^<-8>M）通过成肌细胞融合导致肌管显著增加。(2) MTF的分离鉴定。在胎儿肌肉、肺和肾的SFE中检测到MTF活性。通过凝胶过滤，检测了3-10kD和10-30kD组分的活性。用肝素亲和层析0.9 ~ 1.2 m NaCI溶液洗脱MTF，鉴定为与IGF-1不同的单蛋白带。(3) MTF对牛成肌细胞AchE和AchR表达的影响。在G_0期分化的牛成肌细胞上表达AchE和AchR。MTF和IGF-1诱导牛成肌细胞AchE和AchR的表达。(4)克隆牛成肌细胞的建立。克隆牛成肌细胞Bmb-1和Bmb-2在牛成肌细胞菌落培养中建立。用抗去精蛋白抗体染色，形成肌管。MTF和IGF-1可刺激Bmb-2克隆细胞的肌管形成。(5)原代培养的牛成肌细胞形成三维组织。将琼脂板上的成肌细胞聚集成松散团块的细胞聚集体转移到Culstir烧瓶中，以70 rpm左右的转速进行培养。肿块致密性逐渐增大，形成大小不一（直径250 ~ 500 μ m）的多细胞球体。MTF和IGF-1加速了球体的形成。结果表明，在培养体系中，牛成肌细胞可以重建三维结构。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