Milasincic D J, Dhawan J, Farmer S R
Department of Biochemistry, Boston University Medical School, Massachusetts 02118, USA. .
In Vitro Cell Dev Biol Anim. 1996 Feb;32(2):90-9. doi: 10.1007/BF02723040.
Our previous studies have demonstrated that expression of growth-associated genes is regulated by the adhesive state of the cell. To understand the role of cell adhesion in regulating the switch from growth to differentiation, we are studying the differentiation of mouse myoblasts into multinucleated contractile myotubes. In this report, we describe a novel means of culturing C2C12 myoblasts that permits an analysis of the role of cell adhesion in regulating the sequential induction of muscle-specific genes that control myogenesis. Suspension of an asynchronous, proliferating population of myoblasts in a viscous gel of methylcellulose dissolved in medium containing 20% serum induces growth arrest in G0 phase of the cell cycle without a concomitant induction of muscle-specific genes. Reattachment to a solid substratum in 20% serum, 0.5 nM bFGF, or 10 nM IGF-1 rapidly activates entry of the quiescent cells into G1 followed by a synchronous progression of the cell population through into S phase. bFGF or IGF-1 added separately facilitate only one passage through the cell cycle, whereas 20% serum or the two growth factors added together support multiple cell divisions. Adhesion of suspended cells in DMEM alone or with 3 nM IGF-1 induces myogenesis as evidenced by the synthesis of myogenin and myosin heavy chain (MHC) proteins followed by fusion into myotubes. bFGF completely inhibits this differentiation process even in the presence of myogenic doses of IGF-1. Addition of 3 nM IGF-1 to quiescent myoblasts maintained in suspension culture in serum-free conditions does not induce myogenin or MHC expression. Thus, adhesion is a requirement for the induction of muscle gene expression in mouse myoblasts. The development of a muscle cell culture environment in which proliferating myoblasts can be growth arrested in G0 without activating muscle-specific gene expression provides a means of analyzing the synchronous activation of either the myogenic or growth programs and how adhesion affects each process, respectively.
我们之前的研究表明，生长相关基因的表达受细胞黏附状态的调控。为了解细胞黏附在调节从生长到分化转变过程中的作用，我们正在研究小鼠成肌细胞向多核收缩性肌管的分化。在本报告中，我们描述了一种培养C2C12成肌细胞的新方法，该方法能够分析细胞黏附在调节控制肌生成的肌肉特异性基因顺序诱导中的作用。将异步增殖的成肌细胞群体悬浮于溶解在含20%血清培养基中的甲基纤维素粘性凝胶中，可诱导细胞周期停滞于G0期，而不会同时诱导肌肉特异性基因。在20%血清、0.5 nM碱性成纤维细胞生长因子（bFGF）或10 nM胰岛素样生长因子-1（IGF-1）中重新附着于固体基质，可迅速激活静止细胞进入G1期，随后细胞群体同步进入S期。单独添加bFGF或IGF-1仅促进细胞周期的一次通过，而20%血清或两种生长因子一起添加则支持多次细胞分裂。悬浮细胞单独在DMEM中或与3 nM IGF-1一起黏附可诱导肌生成，这可通过肌细胞生成素和肌球蛋白重链（MHC）蛋白的合成以及随后融合成肌管来证明。即使存在成肌剂量的IGF-1，bFGF也完全抑制这种分化过程。向无血清条件下悬浮培养的静止成肌细胞中添加3 nM IGF-1不会诱导肌细胞生成素或MHC表达。因此，黏附是小鼠成肌细胞中诱导肌肉基因表达的必要条件。开发一种肌肉细胞培养环境，其中增殖的成肌细胞可在G0期生长停滞而不激活肌肉特异性基因表达，为分析成肌或生长程序的同步激活以及黏附如何分别影响每个过程提供了一种方法。
