Ernst C W, McFarland D C, White M E
Department of Animal Science, Ohio Agricultural Research and Development Center, Columbus, USA.
Differentiation. 1996 Oct;61(1):25-33. doi: 10.1046/j.1432-0436.1996.6110025.x.
Myogenic satellite cells are essential for the development of postnatal skeletal muscle. The proliferation and differentiation of these cells are, in part, regulated by the insulin-like growth factors (IGFs), and it has been shown that the IGF binding proteins (IGFBPs) are capable of modulating the actions of IGFs. We have examined the endogenous expression of IGF-II, IGFBP-2 and the myogenic regulatory factor, myogenin, during differentiation of clonally derived turkey muscle satellite cells. Cells were harvested at approximately 80% of confluent density. Additional cultures were rinsed, fed differentiation medium and harvested when approximately 20%, 60% and 80% differentiated (fused). Northern blot analyses were performed using total cellular RNA and labeled rat cDNAs specific for IGF-II, IGFBP-2 and myogenin. A single IGF-II mRNA transcript of approximately 4.0 kb was observed. The relative mRNA abundance was highest in proliferating cultures and decreased with the onset of differentiation, to approximately 60% of initial levels where it remained throughout differentiation. Use of the IGFBP-2 cDNA probe indicated a single mRNA transcript of approximately 2.0 kb. The level of expression of IGFBP-2 mRNA was highest in proliferating cells and decreased to 25%, 16% and 11% of initial levels as differentiation progressed. A single 1.8 kb mRNA transcript was detected with the myogenin probe. Expression of myogenin was undetectable in proliferating cultures and increased significantly as differentiation progressed. Serum-free medium was conditioned for 24 h (CM) at each time point and collected from similar cultures. An IGFBP species of M(r) approximately 30,000 was detected in CM by probing western blots with [125I] IGF-I (ligand blot analysis). The intensity of this band decreased with differentiation to 35%, 24% and 18% of the level for proliferating cultures. Western blots were also probed with an antibody raised against the M(r)-34,000 bovine IGFBP-2. This antibody specifically bound to the M(r)-30,000 IGFBP, and the level of antibody binding decreased as differentiation progressed. It therefore appears that IGF-II, IGFBP-2 and myogenin are expressed in a differentiation-dependent manner by turkey myogenic satellite cells and may thus be involved in the process of differentiation of avian muscle cells.
生肌卫星细胞对于出生后骨骼肌的发育至关重要。这些细胞的增殖和分化部分受胰岛素样生长因子（IGFs）调控，并且已经表明胰岛素样生长因子结合蛋白（IGFBPs）能够调节IGFs的作用。我们研究了克隆来源的火鸡肌肉卫星细胞分化过程中IGF-II、IGFBP-2和生肌调节因子肌细胞生成素的内源性表达。细胞在汇合密度约80%时收获。另外的培养物冲洗后，加入分化培养基，在约20%、60%和80%分化（融合）时收获。使用总细胞RNA和针对IGF-II、IGFBP-2和肌细胞生成素的标记大鼠cDNA进行Northern印迹分析。观察到一条约4.0 kb的单一IGF-II mRNA转录本。相对mRNA丰度在增殖培养物中最高，并随着分化开始而降低，降至初始水平的约60%，并在整个分化过程中保持该水平。使用IGFBP-2 cDNA探针显示一条约2.0 kb的单一mRNA转录本。IGFBP-2 mRNA的表达水平在增殖细胞中最高，并随着分化进展降至初始水平的25%、16%和11%。用肌细胞生成素探针检测到一条1.8 kb的单一mRNA转录本。在增殖培养物中未检测到肌细胞生成素的表达，并且随着分化进展显著增加。在每个时间点，无血清培养基在类似培养物中培养24小时（条件培养基，CM）并收集。通过用[125I]IGF-I探测Western印迹（配体印迹分析）在CM中检测到一种分子量约为30,000的IGFBP。该条带的强度随着分化降低至增殖培养物水平的35%、24%和18%。还用针对分子量为34,000的牛IGFBP-2产生的抗体探测Western印迹。该抗体特异性结合分子量为30,000的IGFBP，并且抗体结合水平随着分化进展而降低。因此，似乎IGF-II、IGFBP-2和肌细胞生成素由火鸡生肌卫星细胞以分化依赖性方式表达，因此可能参与禽类肌肉细胞的分化过程。
