骨骼肌微损伤自体修复中超微结构变化与蛋白质组学研究
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摘要
目的:从蛋白质组代谢角度分析骨骼肌微损伤自体修复过程中不同功能蛋白质组与肌纤维超微结构恢复的同步变化机制。方法:Wistar雄性大鼠36只随机分为安静对照组(D组)、运动后即刻组(0 h组)、运动后24 h组(24 h组)、运动后48 h组(48 h组)、运动后72 h组(72 h组)及运动后168 h组(168 h组)每组6只。运动方案:跑台下坡跑,坡度-16度,速度18 m/min,运动持续60 min,运动后相应时间处死。超薄切片、染色,用JEM-1200EX透射电镜分析不同组别骨骼肌的超微结构。以D组电泳图谱作参考,进行特异蛋白质表达量分析并对表达受到激活或者抑制的蛋白质进行进一步质谱鉴定。特异蛋白质按照功能不同分为I、II、III和IV类。结果:运动后即刻,参与细胞损伤修复的蛋白质(II类)包括膜联蛋白A5、维生素D结合蛋白、α1抗胰蛋白酶前体、DJ-1蛋白、免疫球蛋白、热休克蛋白β1、着丝粒关联蛋白E表达先于超微结构变化;运动后24~72 h,损伤修复过程中能量代谢相关蛋白质(I类)如辅酶Q9、烟酰胺腺嘌呤二核苷酸脱氢酶、乙酰基转移酶、异柠檬酸脱氢酶、磷酸甘油-3脱氢酶受到显著激活,可能促进了线粒体自身修复进而加快肌纤维再生速度;运动后48 h组和72 h组内Trim72蛋白、角蛋白、核糖体蛋白、肌球蛋白、载脂蛋白和内质网驻地蛋白29参与骨骼肌构成的结构蛋白质(III类)表达出现显著上升,这与超微结构恢复具有一定的同步性。结论:骨骼肌微损伤后0~48 h之内细胞损伤修复蛋白表达先于超微结构变化;在损伤修复后24~72 h之内线粒体酶系统及能量代谢相关蛋白表达先于超微结构修复,这可能促进了线粒体自身修复进而加快肌纤维再生速度;在损伤修复后24~48 h之内,骨骼肌结构蛋白表达与超微结构恢复具有一定的同步性。
Objective To study the synchronous mechanism between proteomics with different functions and change in ultrastructure in the process of self-repairing of skeletal muscle micro-injury. Methods 36 male Wistar rats were randomly divided into normal sedentary group,immediate after exercise group,24 hours,48 hours,72 hours and 168 hours after exercise groups. Rats in exercise groups ran downhill on a treadmill with-16 degrees at the speed of 18 m/min for 60 min. The change in ultrastructure of skeletal muscle was observed by transmission electron microscope,and specific proteins were divided into class I,II,III and IV based on their functions. Results Immediately after exercise,the expression of class II proteins,including annexin A5,vitamin D-binding protein,α1-antitrypsin precursor,protein DJ-1,immunoglobulin,heat shock protein β1,and centromere-associated protein E preceded the ultrastructural changes of skeletal muscle. From24 to 72 hours after exercise,the class I proteins,including coenzyme Q9,NADH dehydrogenase,dihyd-rolipoyllysine-residue acetyltransferase,isocitrate dehydrogenase,and glycerin-3 phosphate dehydrogenase were activated significantly. From 48 to 72 hours after exercise,excessive expression of class III proteins,including tripartite motif-containing protein 72,keratin,ribosomal protein,myosin,apolipoprotein and endoplasmic reticulum resident protein 29 were observed. Conclusion Class II proteins were expressed before the ultrastructural changes in skeletal muscle cells within 0 to 48 hours after exercise. From 24 to 72 hours after exercise,class I proteins expression preceded the ultrastructural restoration of skeletal muscle. And within 24 to 48 hours after exercise,class III proteins expression were synchronized with the ultrastructural recovery of skeletal muscle.
Objective To study the synchronous mechanism between proteomics with different functions and change in ultrastructure in the process of self-repairing of skeletal muscle micro-injury. Methods 36 male Wistar rats were randomly divided into normal sedentary group,immediate after exercise group,24 hours,48 hours,72 hours and 168 hours after exercise groups. Rats in exercise groups ran downhill on a treadmill with-16 degrees at the speed of 18 m/min for 60 min. The change in ultrastructure of skeletal muscle was observed by transmission electron microscope,and specific proteins were divided into class I,II,III and IV based on their functions. Results Immediately after exercise,the expression of class II proteins,including annexin A5,vitamin D-binding protein,α1-antitrypsin precursor,protein DJ-1,immunoglobulin,heat shock protein β1,and centromere-associated protein E preceded the ultrastructural changes of skeletal muscle. From24 to 72 hours after exercise,the class I proteins,including coenzyme Q9,NADH dehydrogenase,dihyd-rolipoyllysine-residue acetyltransferase,isocitrate dehydrogenase,and glycerin-3 phosphate dehydrogenase were activated significantly. From 48 to 72 hours after exercise,excessive expression of class III proteins,including tripartite motif-containing protein 72,keratin,ribosomal protein,myosin,apolipoprotein and endoplasmic reticulum resident protein 29 were observed. Conclusion Class II proteins were expressed before the ultrastructural changes in skeletal muscle cells within 0 to 48 hours after exercise. From 24 to 72 hours after exercise,class I proteins expression preceded the ultrastructural restoration of skeletal muscle. And within 24 to 48 hours after exercise,class III proteins expression were synchronized with the ultrastructural recovery of skeletal muscle.
引文
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[14]金其贯,刘霞,李淑艳,等.反复离心运动对大鼠骨骼肌损伤和蛋白质降解机制的影响[J].体育科学,2010,6:76-80.
[15]Panchangam A,Claflin DR,Palmer ML,et al.Magnitude of sarcomere extension correlates with initial sarcomere length during lengthening of activated single fibers from soleus muscle of rats[J].Biophys J,2008,95(4):1890-1901.
[16]Morgan DL,Proske U.Popping sarcomere hypothesis explains stretch-induced muscle damage[J].Clin Exp Pharmacol Physiol,2004,31(8):541-545.
[17]Butterfield TA,Herzog W.Magnitude of muscle straindoes not influence serial sarcomere number adaptations following eccentric exercise[J].Pflugers Arch,2006,451(5):688-700.
[18]Yu JG,Carlsson L,Thornell LE.Evidence for myofibril remodeling as opposed to myofibril damage in human muscles with DOMS:An ultrastructural and immunoelectron microscopic study[J].Histochem Cell Biol,2004,121(3):219-227.
[2]Chen TC.Variability in muscle damage after eccentric exercise and the repeated bout effect[J].Res Q Exerc Sport,2006,77(3):362-371.
[3]于新凯,晁唯伟,左群.运动性骨骼肌损伤超微结构变化的研究进展[J].解剖科学进展,2013,19(3):298-302.
[4]张翔,张学林,许寿生.运动与骨骼肌超微结构变化[J].中国运动医学杂志,2010,29(1):109-113.
[5]Mc Phee JS,Williams AG,Perez-Schindler J,et al.Variability in the magnitude of response of metabolic enzymes reveals patterns of co-ordinated expression following endurance training in women[J].Exp Physiol,2011,96(7):699-707.
[6]van den Broek NM,Ciapaite J,Nicolay K,et al.Comparison of in vivo postexercise phosphocreatine recovery and resting ATP synthesis flux for the assessment of skeletal muscle mitochondrial function[J].Am J physiol cell physiol,2010,299:C1136-C1143.
[7]魏源,王革,卓莉.运动性骨骼肌微损伤机制的研究进展[J].上海体育学院学报,2007,31(2):67-73.
[8]朱荣,马延超,王瑞元.一次大强度运动对大鼠骨骼肌泛素蛋白酶体途径基因表达及蛋白质降解的影响[J].中国运动医学杂志,2012,31(5):414-419.
[9]白巍,张海平.离心运动对大鼠骨骼肌线粒体ATP酶活性及desmin表达影响的研究[J].沈阳体育学院学报,2013,32(2):86-88.
[10]葛新发,董贵俊,王玉站,等.重复大强度运动对骨骼肌蛋白质组变异研究[J].体育科学,2013,33(5):41-49.
[11]董贵俊,吕晨曦,葛新发,等.一次和重复大强度离心运动前后大鼠骨骼肌超微结构变化[J].中国运动医学杂志,2013,32(2):167-173.
[12]Bradford MM.A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein dye binding[J].Anal Biochem,1976,72:248-254.
[13]田野,马鹏鹏,郭时杰,等.连续运动后延迟性肌肉损伤的适应性研究[J].中国运动医学杂志,2003,22(2):138-142.
[14]金其贯,刘霞,李淑艳,等.反复离心运动对大鼠骨骼肌损伤和蛋白质降解机制的影响[J].体育科学,2010,6:76-80.
[15]Panchangam A,Claflin DR,Palmer ML,et al.Magnitude of sarcomere extension correlates with initial sarcomere length during lengthening of activated single fibers from soleus muscle of rats[J].Biophys J,2008,95(4):1890-1901.
[16]Morgan DL,Proske U.Popping sarcomere hypothesis explains stretch-induced muscle damage[J].Clin Exp Pharmacol Physiol,2004,31(8):541-545.
[17]Butterfield TA,Herzog W.Magnitude of muscle straindoes not influence serial sarcomere number adaptations following eccentric exercise[J].Pflugers Arch,2006,451(5):688-700.
[18]Yu JG,Carlsson L,Thornell LE.Evidence for myofibril remodeling as opposed to myofibril damage in human muscles with DOMS:An ultrastructural and immunoelectron microscopic study[J].Histochem Cell Biol,2004,121(3):219-227.