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目的:研究糖皮质激素地塞米松(dexamethasone,DEX)对体外培养肌管的形态、蛋白质合成/分解代谢及肌肉萎缩蛋白Fbox-1(Atrogin-1)的表达,Atrogin-1基因沉默是否可减轻肌管萎缩。方法:体外培养小鼠肌纤维细胞株C2C12细胞并分化为肌管后,用DEX处理48h,同位素3H-酪氨酸掺入法检测蛋白合成,3H-酪氨酸释放率检测蛋白分解代谢;荧光显微镜观察肌管形态并拍照;Northern blot检测Atrogin-1mRNA水平;应用小分子干扰RNA片段(siRNA)技术使Atrogin-1基因沉默后,观察DEX作用下肌管形态的改变。结果:DEX5μmol/L作用后,肌管酪氨酸(tyrosine)掺入率下降17.4%,同时tyrosine释放率升高24.7%,使肌管形态变细、萎缩;剂量依赖性地升高Atroign-1蛋白表达水平,使Atroign-1mRNA表达升高3倍;Atrogin-1基因沉默可改善DEX引起的肌管萎缩。结论:DEX促进肌管蛋白质分解代谢,并抑制蛋白合成代谢,导致肌肉萎缩;Atrogin-1基因沉默可改善DEX引起的肌肉萎缩,Atrogin-1基因可能是逆转肌肉消耗性营养不良的有效靶点。
OBJECTIVE: To study the effects of dexamethasone (DEX) on the morphology of myotubes, the protein synthesis / catabolism and the expression of Atrogin-1 (Atrogin-1) in vitro and whether the Atrogin-1 gene silencing can be alleviated Myotube atrophy. Methods: The mouse myofibroblasts C2C12 cells were cultured in vitro and differentiated into myotubes. The cells were treated with DEX for 48h. The 3H-tyrosine incorporation assay was used to detect the protein synthesis. 3H-tyrosine release assay was used to detect the protein catabolism. Fluorescence microscopy The morphology of myotubes was observed and photographed. The Atrogin-1 mRNA level was detected by Northern blot. The Atrogin-1 gene was silenced by small interfering RNA (siRNA) technique. The morphological changes of myotubes under DEX were observed. RESULTS: After treated with DEX 5 μmol / L, the tyrosine incorporation decreased by 17.4% and the tyrosine release rate increased by 24.7%. The morphology of myotubes became thin and atrophied. Atroign-1 Protein expression level, the Atroign-1mRNA expression increased 3-fold; Atrogin-1 gene silencing can improve DEX induced myotube atrophy. CONCLUSION: DEX can promote catabolism of myotubes and inhibit protein synthesis and metabolism, leading to muscle atrophy. Atrogin-1 gene silencing can improve the muscle atrophy caused by DEX. Atrogin-1 gene may be an effective target to reverse muscular dystrophy.