Department of Animal Biotechnology, College of Animal Life Sciences, Kangwon National University, Chuncheon, South Korea.
In Vitro Cell Dev Biol Anim. 2012 Mar;48(3):175-85. doi: 10.1007/s11626-012-9484-1. Epub 2012 Jan 21.
Studies on skeletal muscle cell specification and development have demonstrated in the past that calpains interact with various transcriptional factors in regulating the cellular function. It has therefore, been assumed that transcriptional factors like myogenin, MyoD, Myf5, and MRF4 that are active during the myogenic differentiation might be affected and degraded by calpains. Therefore, to examine the biochemical adaptations of myoblasts during myocyte formation and muscle development comprehensively, the current study was designed to identify the effect of calpeptin (calpain inhibitors) on protein expression during differentiation of C2C12 mouse myoblast. Cells were proliferated to near 80% confluence under Dulbecco's modified eagle medium and differentiated further in 2% HS with 50 μM calpeptin. Incubated cells were collected at 0, 12, and 72 h and later the cell proteins were focused onto pH 4-7 IEF strip, followed by 12.5% SDS-PAGE. Obtained spots on the gels were compared and matched using commercial 2-DE analysis software and matched spots were identified by MALDI-ToF and/or Q-Tof systems. Conclusively, cell differentiation was observed to be active from 12 to 72 h however, calpeptin affected the differentiation process and cut down the rate of fusion by approximately 50%. Out of 41 proteins identified, 12 proteins were found to be upregulated where as 29 proteins were downregulated.
过去的研究表明，在骨骼肌细胞的特化和发育过程中，钙蛋白酶与各种转录因子相互作用，调节细胞功能。因此，人们假设在肌生成分化过程中活跃的转录因子，如肌球蛋白、MyoD、Myf5 和 MRF4，可能会受到钙蛋白酶的影响和降解。因此，为了全面研究成肌细胞形成和肌肉发育过程中肌细胞的生化适应，本研究旨在确定钙肽素（钙蛋白酶抑制剂）对 C2C12 小鼠成肌细胞分化过程中蛋白表达的影响。细胞在 Dulbecco 改良 Eagle 培养基中增殖至接近 80%的汇合度，然后在 2% HS 中进一步分化，加入 50 μM 钙肽素。在 0、12 和 72 h 时收集孵育细胞，然后将细胞蛋白聚焦到 pH 4-7 IEF 条上，再进行 12.5% SDS-PAGE。使用商业 2-DE 分析软件比较和匹配凝胶上的斑点，并通过 MALDI-ToF 和/或 Q-Tof 系统鉴定匹配的斑点。结论是，细胞分化从 12 到 72 h 都很活跃，然而，钙肽素影响了分化过程，使融合率降低了约 50%。在鉴定的 41 种蛋白质中，有 12 种蛋白质上调，29 种蛋白质下调。
