电刺激对C2C12骨骼肌细胞糖代谢及其机制研究
The Effects of Electrical Stimulation on Glycometabolism and Its Regulating Mechanism in C2C12 Skeletal Muscle Cell
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摘要: 目的:利用培养的骨骼肌细胞作为模型,研究收缩运动对肌糖原、AMPK-α2及GLUT4表达的影响。方法:分化5天的C2C12骨骼肌肌管,电刺激(15V,3Hz)60min,120min后随即收样检测,酶联免疫法测肌管中GLUT4蛋白含量,Western blot检测肌管中AMPK-α2蛋白表达,实时荧光定量PCR测定肌管中GLUT4mRNA及AMPK-α2mRNA的表达。结果:1)糖原含量在刺激120min时与对照组相比显著降低(P<0.05),LDH活性与对照组相比无显著性差异(P>0.05);2)电刺激60min时,GLUT4基因表达和蛋白含量增加(P<0.05),刺激120 min时,GLUT4蛋白含量增加显著(P<0.01);3)电刺激引起AMPK-α2mRNA表达增加(P<0.05),AMPK-α2蛋白表达虽有所增加,却无统计意义(P>0.05)。结论:1)15V,3Hz的电刺激强度,可引起培养的C2C12骨骼肌细胞产生收缩运动,建立研究骨骼肌收缩功能变化的细胞模型;2)长时间电刺激可降低C2C12骨骼肌细胞的肌糖原含量,激活AMPK-α2mRNA和GLUT4的表达。Abstract: Abstract:Objective:The aim of this study was to measure changes in cellular glucose content,AMPK-α2 and GLUT4 gene expression in electrically stimulated mouse C2C12 skeletal muscle cells.Method:After five days of differentiation,myotubes were stimulated using an electrical stimulator set at 15v at a frequency of 3 Hz.Acute stimulations were performed for 60 or 120 min in each dish.GLUT4 protein contents and AMPK-α2 protein expression in myotubes were detected respectively using Enzyme-Linked ImmunoSorbent Assay(ELISA)and Western blot.GLUT4 mRNA and AMPK-α2 mRNA in myotubes were detected using fluorescence quantification PCR technology.Results:1) Compared with the control group,the glucose contents of 120 min group was significantly reduced(P<0.05) and the activity of LDH was no significant difference(P>0.05).2) GLUT4 gene expression and protein content increased after 60min of electrical stimulation(ES)(P<0.05).3) AMPK-α2 gene expression increased after 60min or 120 min of electrical stimulation(P<0.05),but AMPK-α2 protein expression showed no significant deviation between ES and control group.Conclusion:1)Electrical stimulation can cause cultured C2C12 skeletal muscle cells to produce contraction at the correct frequency(3Hz) using voltages as low as 15V.Therefore we established the cell model of the research skeletal muscle contraction function.2) Long time electrical stimulation can reduce glucose content,activate AMPK-α2mRNA expression,and alter GLUT4 mRNA expression and GLUT4 protein contents in cultured C2C12 skeletal muscle cells.