Faculty of Science and Engineering, Institute for Biomedical Research into Human Movement and Health, Manchester Metropolitan University, Manchester, UK.
J Cell Physiol. 2010 Oct;225(1):240-50. doi: 10.1002/jcp.22252.
Reduced muscle mass and increased susceptibility to TNF-induced degradation accompany inflamed ageing and chronic diseases. Furthermore, C(2) myoblasts display diminished differentiation and increased susceptibility to TNF-alpha-induced cell death versus subcloned C(2)C(12) cells, providing relevant models to assess: differentiation (creatine kinase), growth (protein), death (trypan-blue) and anabolic/catabolic parameters (RT-PCR) over 72 h +/- TNF-alpha (20 ng ml(-1)). At 48 and 72 h, respectively, larger myotubes and significantly higher CK activity (320.26 +/- 6.82 vs. 30.71 +/- 2.5, P < 0.05; 544.94 +/- 27.7 vs. 39.4 +/- 3.37 mU mg ml(-1), P < 0.05), fold increases in myoD (21.45 +/- 3.12 vs. 3.97 +/- 1.76, P < 0.05; 31.07 +/- 3.1 vs. 6.82 +/- 1.93, P < 0.05) and myogenin mRNA (241.8 +/- 40 vs. 36.80 +/- 19.3, P < 0.05; 440 +/- 100.5 vs. 201.1 +/- 86, P < 0.05) were detected in C(2)C(12) versus C(2). C(2)C(12) showed significant increases in IGF-I mRNA (243.05 +/- 3.87 vs. 105.75 +/- 21.95, P < 0.05), reduced proliferation and significantly lower protein expression (1.21 +/- 0.28 vs. 1.79 +/- 0.29 mg ml(-1), P < 0.05) at 72 h versus C(2) cells. Significant temporal reductions in C(2)C(12) IGFBP2 mRNA (28.02 +/- 15.44, 13.82 +/- 8.07, 6.92 +/- 4.37, P < 0.05) contrasted increases in C(2)s (4.31 +/- 3.31, 13.02 +/- 9.92, 82.9 +/- 58.9, P < 0.05) at 0, 48 and 72 h, respectively. TNF-alpha increased cell death in C(2)s (2.67 +/- 1.54%, 34.42 +/- 5.39%, 29.71 +/- 5.79% (0, 48, 72 h), P < 0.05), yet was without effect in C(2)C(12)s at 48 h but caused a small significant increase at 72 h (9.88 +/- 4.02% (TNF-alpha) vs. 6.17 +/- 0.749% (DM), 72 h). TNF-alpha and TNFRI mRNA were unchanged; however, larger reductions in IGF-I (8.2- and 7.5-fold vs. 4.5- and 4.1-fold (48, 72 h)), IGF-IR (2-fold vs. no-significant reduction (72 h)) and IGFBP5 (3.24 vs. 1.38 (48 h) and 2.21 vs. 1.71 (72 h), P < 0.05) mRNA were observed in C(2) versus C(2)C(12) with TNF-alpha. This investigation provides insight into regulators of altered basal hypertrophy and TNF-induced atrophy, providing a model for future investigation into therapeutic initiatives for ageing/wasting disorders.
肌肉质量减少和对 TNF 诱导的降解的易感性伴随着炎症性衰老和慢性疾病而增加。此外，C(2)成肌细胞显示出分化减少和对 TNF-α诱导的细胞死亡的易感性增加，与亚克隆的 C(2)C(12)细胞相比，提供了相关的模型来评估：分化（肌酸激酶）、生长（蛋白质）、死亡（台盼蓝）和合成代谢/分解代谢参数（RT-PCR）在 +/- TNF-α（20ng/ml）下 72 小时。在 48 和 72 小时时，分别观察到更大的肌管和显著更高的 CK 活性（320.26 +/- 6.82 与 30.71 +/- 2.5，P < 0.05；544.94 +/- 27.7 与 39.4 +/- 3.37 mU mg ml(-1)，P < 0.05），肌源性分化因子（myoD）（21.45 +/- 3.12 与 3.97 +/- 1.76，P < 0.05；31.07 +/- 3.1 与 6.82 +/- 1.93，P < 0.05）和肌生成素 mRNA（241.8 +/- 40 与 36.80 +/- 19.3，P < 0.05；440 +/- 100.5 与 201.1 +/- 86，P < 0.05）的 fold-increase 在 C(2)C(12) 中比 C(2)高。在 C(2)C(12) 中，IGF-I mRNA（243.05 +/- 3.87 与 105.75 +/- 21.95，P < 0.05）显著增加，增殖减少，蛋白质表达水平显著降低（1.21 +/- 0.28 与 1.79 +/- 0.29 mg ml(-1)，P < 0.05），在 72 小时与 C(2)细胞相比。C(2)C(12) IGFBP2 mRNA 的时间依赖性降低（28.02 +/- 15.44，13.82 +/- 8.07，6.92 +/- 4.37，P < 0.05），而 C(2) 的增加（4.31 +/- 3.31，13.02 +/- 9.92，82.9 +/- 58.9，P < 0.05）在 0、48 和 72 小时分别观察到。TNF-α增加了 C(2)s 的细胞死亡（2.67 +/- 1.54%，34.42 +/- 5.39%，29.71 +/- 5.79%（0，48，72 小时），P < 0.05），但在 48 小时时对 C(2)C(12)没有影响，但在 72 小时时导致一个小的显著增加（9.88 +/- 4.02%（TNF-α）与 6.17 +/- 0.749%（DM），72 小时）。TNF-α和 TNFRI mRNA 没有变化；然而，IGF-I（8.2- 和 7.5- 倍），IGF-IR（2- 倍）和 IGFBP5（3.24 与 1.38（48 小时）和 2.21 与 1.71（72 小时）的 mRNA 减少更大，与 C(2)相比，在 C(2)中观察到 C(2)C(12)中（48 小时）和 2.21 与 1.71（72 小时），P < 0.05）。本研究为基础肥大和 TNF 诱导的萎缩的改变提供了调节因子的深入了解，为衰老/消耗性疾病的治疗方法提供了模型。
