目的:采用蛋白组学技术研究6周有氧跑台运动对C57BL/6小鼠骨骼肌蛋白表达的影响.通过筛选对比安静组与运动组小鼠骨骼肌差异表达蛋白,分析有氧运动引发的骨骼肌代谢相关信号通路变化,探讨运动增进健康、防治疾病的分子机理.方法:选用C57BL/6雄性小鼠40只,将其随机分为正常饮食组(NC,n=20)和正常饮食运动组(NE,n=20),均饲以基础饲料.NE组进行6周强度为75％VO2max的跑台运动.6周后提取小鼠股四头肌总蛋白,采用Bradford法检测蛋白浓度后进行蛋白质双向电泳(2-DE),采用ImageMaster 2D Platinum V5.0软件分析2-DE电泳图谱,并对差异表达蛋白点采用基质辅助激光解吸附离子化飞行时间质谱( MALDI-TOF-MS)和高效液相色谱一串联谱法(LC-MS/MS)进行分析、采用Mascot检索软件结合NCBI nr数据库鉴定差异表达蛋白,并运用相关分析软件对所得数据进行统计学处理.结果:经6周有氧跑台运动后,比较NC组与NE组小鼠骨骼肌蛋白表达图谱,以蛋白表达量变化1.5倍以上为显著性判定标准,共筛选出32个差异表达蛋白,排除高表达丰度蛋白背景信号后共选出6个差异表达蛋白.其中,与线粒体能量代谢相关的蛋白有3个,分别为呼吸链复合体I (Nadh-Ubiquinone Oxidoreductase 18 Kda Ip Subunit),表达增加2.92倍,线粒体膜电压依赖阴离子通道1和2(Voltage-dependent Anion Channel,VDAC),表达上调1.82倍和3.51倍；与细胞膜修复相关的蛋白Trim72(Tripartite Motif-Containing Protein 72)表达增加2.10倍;糖代谢相关蛋白乳酸脱氢酶(L-Lactate Dehydrogenase B Chain,LDH-B)表达上调1.85倍；Ester Hydrolase C11Orf54Homolog蛋白表达下降56％.结论:6周有氧运动引起小鼠骨骼肌32个蛋白表达显著改变,其中,与线粒体能量代谢相关蛋白、细胞膜修复相关蛋白、糖代谢相关蛋白等均受到影响.由于这些蛋白功能与骨骼肌能量代谢密切关系,提示有氧运动对促进骨骼肌能量代谢、预防代谢相关疾病具有重要作用.%Objective The aim of this study was to explore the molecular mechanisms involved in the effect of aerobic exercise on the protein expression in skeletal muscle of mice. Methods Forty male C57BL/6 mice were randomly divided into control group (NO and exercise group (NE) , and both groups were fed with normal forage. Group NE was forced to run on a treadmill for 6 weeks with the intensity of 75%VO2max. Total proteins were extracted from quadriceps femoris after 6-week exercise, and then separated by two-dimensional gel electrophoresis (2-DE) , and the differentially expressed proteins were analyzed using ImageMaster 2D Platinum V5.0, matrix-assisted laser desorption/ioniza-tion time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem mass spectrometry (LC/LC-MS/MS ) , and finally the differentially expressed proteins were identified using Mascot software and NCBI nr database. A protein expression was considered to be significantly differential ( over-expressed or under-expressed) if the ratio of intensity between two groups was higher than 1.5-fold or lower than0.67. Results After 6-week aerobic exercise, 32 protein spots were found in group NE compared with group NC, in which 3 proteins were up-regulated and 3 were down-regulated after excluding the high abundant proteins. 3 proteins involved in mitochondrial energy metabolism, I.e., Nadh-ubiquinone oxidoreductase 18 Kda Ip subunit (increased 2.92 fold) and voltage-dependent anion channel 1 and 2 (VDAC1, VDAC2) (decreased 0.42 fold and 0.58 fold, respectively) . Tripartite motif-containing . Protein 72 (Trim72) was found to be involved in cell membrane repair machinery (increased 2.10 fold) . L-lactate dehydrogenase B chain (LDH-B) was related to the glucose metabolism (increased 1.85 fold) . Ester hydrolase CllOrf54 homolog decreased 0.44 fold. Conclusion After 6-week aerobic exercise, 32 differentially expressed proteins were identified which involved in energy metabolism, repairing cell membrane and glucose metabolism. Our data indicate that aerobic exercise plays a key role in skeletal muscle energy metabolism and in preventing metabolic diseases.
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