摘要
目的:本研究运用生命科学领域中前沿的比较蛋白质组学理论、方法与技术,探讨在运动心脏重塑过程中心房肌蛋白质组差异性表达的动态变化,筛选出有意义的心房肌目标蛋白质,全面、系统的诠释运动心脏的发生、发展规律,为研究运动心脏及慢性心血管疾病的运动康复提供实验依据和新的思路与方法。
方法:以54只雄性SD大鼠为研究对象,按体重随机分为6组,其中实验组3组(分为4周、8周与12周运动组),对照组3组(分为4周、8周和12周平行对照组),实验组采用12周中等强度(70～80%VO_2max)递增运动负荷跑台训练构建运动心脏实验动物模型,分别于运动4、8、12周后与平行对照组同时麻醉处死,计量心重量,提取心房肌蛋白样品,采用双向凝胶电泳技术建立实验组与对照组蛋白质组的差异性表达图谱;选择运动后差异表达量≥10倍的蛋白质点作为备选的目标蛋白质,采用串联飞行时间质谱仪进行肽质量指纹分析后,选取一级质谱图上峰值较高的肽段进行二级质谱确证。
结果与结论:经4、8、12周运动后,与对照组相比实验组心脏重量指数分别增加18.5%、34.5%与36.6%,均具有显著性差异(p＜0.05),出现运动心脏的形态学变化。在实验组心房肌蛋白质差异表达量变化≥10倍的38个蛋白质点中,经质谱鉴定、确证3个备选的目标蛋白质点。心钠素前体为4周实验组表达量上调10倍的蛋白质,其表达量的升高提示:可能与抑制心肌细胞的过度生长及血压的调节有关;热休克蛋白β_2和热休克蛋白60为12周实验组表达量分别上调50和100倍的点,均属于热休克蛋白家族,它们表达量升高提示:长期中等强度的耐力运动有利于心肌细胞保护和增强心肌功能,预防心肌在各种应激情况下的损伤。且热休克蛋白还可能直接参与心肌肥大的发生。在运动心脏的发生过程中,大鼠心房肌蛋白质组发生的差异性变化与心脏形态、结构的重塑及其调节、心房内分泌、心血管系统功能的改善及应激的适应有关。
Intention: using the advanced theory and technology of comparative Proteomic in life sciences field, we discuss the dynamic change of atrial muscle Proteomic differential expressive in athletic heart remodeling process, and screen out correlative target protein, open out the happening and development rules of the athletic heart roundly and systematically, afford experimentation gist and new thought and means for studying athletic heart and chronic cardiovascular disease sport rehabilitation.
Method: 54 male SD rats were divided into six groups randomly according to their weight: 3 exercise groups and 3 rest groups. The exercise groups was sent to 12 weeks progressive increasing load volume training of middling intensity (70-80%VO_2max). The exercise groups and the rest groups executed after anesthetize at the same time after 4、8 and 12 weeks exercise respectively, the cordis weight was figured out, the auricular muscle was extracted and then to build the exercise groups and the rest groups' s proteome differential expression profile by two-dimensional gel electrophoresis(2-DE); selected protein spots of differential expression volume≥10 folds as object protein, the higher peak value peptide was carried through mass spectrometry identification with EI-MS/MS after peptide mass fingerprint analysis by ULGRAFL-FLEX-TOF/TOF.
Results and conclusion: the heart weight index of the exercise groups increased 18. 5%、34.5% and 36. 6% respectively after 4, 8, 12 weeks exercise and were significantly higher than those in control groups(p<0. 05), and the changes of athletic cardiac morphological were observed. Identified three object protein spots by mass spectrum from 38 protein spots which the quantitative change of the exercise groups auricular muscle differential expression volume≥10 folds. Atrial natriuretic factor precursor (proANF) was the protein that expression increased 10 folds after four weeks exercise. Hints: the expression increase of the proANF is relative to restraining the over much growth of the cardiac muscle cell and adjusting the blood pressure; Heat shock protein beta-2(HSPβ_2) and 60kDa heat shock protein(HSP60) were the proteins that expression increased 50 and 100 folds after twelve weeks exercise, they are belong to the heat shock protein family. Their increased of the expression hint: Long-term medium intensity aerobic exercise is propitious to protecting cardiac muscle cell and strengthen its function, prevent from myocardial damage under the various condition that responding to stimulation. And the expression increased of the HSP hint that it is concerned with the happening of myocardial obesity. The differential changes of auricular muscle Proteomic were correlated with remodeling of cordis shape and structure and its control, improving atria incretion and cardiovascular system function, and the adaptation of responding to stimulation in athletic heart remodeling process.
引文
[1]张浩.运动员心脏与运动[J].中国临床康复,2005,9(36):108-109
[2]浦钧宗.运动心脏的回顾、现状和瞻望[J].体育与科学,1999,20(120):4-8
[3]常芸.运动心脏重塑的调节[J].体育科学,2004,24(2):12-16
[4]常芸,林福美,孙迎.运动心脏的自身调节[J].体育科学,1998,18(1):67-72
[5]常芸,赵中应,孙迎.耐力训练后大鼠心肌中原癌基因c-myc的表达[J].中国运动医学杂志,1999,18(2):111-114
[6]常芸,孙迎,陈小同,郭军.运动心脏内分泌功能可复性的研究[J].中国运动医学杂志,1999,18(1):3-6
[7]Baldwin KM.Research in the exercise sciences:Where do we go from here? J Appl Physiol,2000,88:332-336
[8]Yuan O,Fontenele-Neto JD,Fricker LD.Effect of voluntary exercise on genetically obese Cpefat/fat mice:quantitative proteomics of serum.Obes Res.2004 Jul,12(7):1179-1188
[9]Rogers D S,Li Z B,Randall M J,et al.Identification of Candidate Proteins for the Cardioprotective Effects of Exercise.Med Sci Sports Exe.[J]2003,35(5):367
[10]Boluyt Mo,Brevick J L.et al.Changes in the rat heart proteome induced by exercise training:Increased abundance of heat shock protein hsp20[J].Proteomics,2006,6(10):3154-69
[11]史绍蓉,王步标,余玲,等.蛋白质组与蛋白质组学及其在运动医学领域中的应用[J].中国运动医学杂志,2005,24(5):632-633
[12]张松江,史绍蓉.急性大强度力竭运动大鼠骨骼肌比较蛋白质组学研究[J].体育科学,2006,26(5):59-63
[13]张松江,史绍蓉.急性力竭运动大鼠骨骼肌蛋白质组差异性表达的研究[J].中国运动医学杂志,2006,25(5):56-158
[14]沙继斌,刘明海.急性力竭性运动对大鼠淋巴细胞蛋白质组影响的初步研究[J].山东体育学院学报,2005,21(3):62-64
[15]史绍蓉,刘田,龚丽,等.力竭运动大鼠心室肌蛋白质组表达特征[J].中国组织工程研究与临床康复,2007,11(43):8691-8697
[16]龚丽.递增运动负荷训练对大鼠心房肌蛋白质组影响的研究:[硕士学位论文].长沙.湖南师范大学,2007
[17]常芸,袁箭峰,祁永梅,等.运动心脏重塑与微损伤发生中的细胞凋亡现象[J].中国运动医学杂志,2003,22(4):344-349
[18]Bradford,M.A Rapid and Sensitive Method for the quantitation of Microgram quantities of Protein utilizing the Principle of Protein Dye Binding.Anal Biochem.1976,72:248-254
[19]Gorga,Obermaierc,Boguthg,et al.The Current State of Two-dimensional Electrophoresis with Immobilized pH Gradients[J].Electrophoresis,2000,21(6):1037-1053
[20]刘田.运动性疲劳大鼠心室肌蛋白质组差异性表达的研究:[硕士学位论文].长沙.湖南师范大学,2007
[21]陈主初,梁宋平.肿瘤蛋白质组学[M].湖南:湖南科学技术出版社,2002:10-22
[22]赵海军,邓树勋.运动心脏及其形成机制(综述)[J].体育学刊,2002,9(2):121-123
[23]常芸,林福美,陈小同,等.运动心脏结构可复性研究[J].中国运动医学杂志,1999,18(3):202-207
[24]高云秋.运动与心脏左房增大[J].中国运动医学杂志,1995,14(3):202-211
[25]彭峰林,乔秀芳.运动与心脏结构的重塑[J].体育科研,2001,22(2):35-36
[26]佟长青,张引国,郭建增,等.运动性和高血压性肥大心脏心肌细胞超微结构的对比研究[J].中国运动医学杂志,2001,20(2):158-166
[27]常芸,林福美,陈小同,等.运动心脏重塑的发生与转归[J].体育科学,2000,20(5):46-50
[28]张松江.急性力竭运动大鼠腓肠肌的比较蛋白质组学研究:[硕士学位论文].长沙.湖南师范大学,2006
[29]刘正冬,李可某,冯建英.大强度游泳抑制小鼠骨骼肌谷氨酸胺合成酶活性[J].体育科学,2001,21(3):62-65
[30]夏家辉.生命科学研究(2003)年中国博士后生命科学学术研讨会暨院士论坛[M].北京科学出版社2003:32-36,67-116
[31]陈金星,张芊,郑翼,等.高血压大鼠心脏细胞骨架蛋白α-微管蛋白和结蛋白[J].高血压杂志2006,14(2):124-128
[32]王蕾,郭子新,席丰,等.肌动蛋白、肌钙蛋白及纤维连接蛋白在心肌缺血中的研究[J].实用心脑肺血管病杂志,2006,14(8):615-617
[33]Vesely D L.Atrial natriuretic peptide prohormone gene expression:hormones and diseases that upregulate its expression[J].IUBMB Life,2002,53(3):153-159
[34]林善琰.当代肾脏病学[M].上海科技教育出版社,2001:83-87
[35]Vesely D L,et al.Clin Fxp Pharm Physiol,1995,22(2):108-114
[36]Vesely D L,et al.Circulation,1994,90(2):1129-1140
[37]Vesely D L,et al.Metabolism,1996,45(3);315-319
[38]曾强,王培勇,唐朝枢,等.心钠素前体分子内调控对心肌Na~+-K~+-ATP酶的作用[J].中国应用生理学杂志,1999,15(2):115-118
[39]孙建波,路新国,张蕾,等.心钠素在医学与运动医学中的研究与进展[J].中国临床康复,2005,9(12):198-199
[40]常芸.运动心脏的实验研究[M].北京:人民体育出版社,1998:56-62
[41]Oliver P M,Fox J E,Kim H S,et al.Hypertension,cardiachy-pertrophy,and sudden death in mice lacking natriuretic peptide receptor A[J].Proc Natl Acad Sci USA,1997,94:14730-14735
[42]Horio T,Nishikimi T,Yoshihara F,et al.Inhibitory regulation of hypertrophy by endogenous atrial natriuretic peptide in cultured cardiac myocytes.Hypertension,2000,35(1Pt1):19-24
[43]潘珊珊,陆爱云,张炎,等.运动对心脏心钠素受体基因表达的影响[J].中国运动医学杂志,2004,23(4):386-389
[44]油红文,陈曦,油红捷,等.心钠素及内皮素对心肌细胞肥大及心脏成纤维细胞增殖的影响[J].高血压杂志,2006,14(5):375-378
[45]Han B,Hasin Y.Cardiovascular effects of natriuretic peptides and their interrelation with endothelin-1[J].Cardiovase Drugs Ther,2003,17:41-52
[46]Bold A J,Ma KK,Zhang Y,et al.The physiological and pathophysiological modulation of the endocrine function of the heart[J].Can J Physiol Pharmacol,2001,79:705-714
[47]郑澜,潘珊珊.运动与心钠素的研究现状与展望[J].成都体育学院学报,2001,27(4):92-96
[48]王诚,王军力.热休克蛋白在运动医学界的研究进展[J].解放军体育学院学报,2004,23(1):99-102
[49]Morimoto R I,Tissieres A,Ceorgopoulos C,et al.Stress protein in biology and medicine[M].CSHL PRESS,1990:1-36
[50]Hendrick J P,Hartl F U.Molecular chaperone function of heat shock proteins[J].Annu Rev Biochem,1993,62(2):349-384
[51]石娇,于维军,胡兰,等.热休克蛋白的生物学功能研究进展[J].中国畜牧兽医, 2005,32(5):6-7
[52]梁向阳,姚鸿恩,段立公.急慢性运动与骨骼肌热休克蛋白的表达[J].兰州大学学报(医学版),2005,31(2):106-109
[53]王莉萍.热休克蛋白与心肌保护[J].国外医学·生理、病理科学与临床分册,2005,25(1):65-67
[54]程文伟,马勇.热休克蛋白对心脏的保护作用[J].山东医药,2005,45(2):77-78
[55]Bukau B,Horwich A L.The HSP70 and HSP60 chaperone machines[J].Cell,1998,92(3):351-66
[56]Xu Z,Horwich AL,Sigler PB.The crystal structure of the asymmetric GroEL-GroES-(ADP)ehaperonin complex[J].Nature,1997,388(4):741-750
[57]龚兴国,于红.热休克蛋白60的研究进展[J].中国病理生理杂志,2004,20(11):2151-2154
[58]Itoh H,Komatsuda A,Ohtani H,et al.Mammalian HSP60 quickly sorted into the mitochondria under conditions of dehdration[J].Eur J Biochem,2002,269(23):5931-5938
[59]郭爱疆,才学鹏.小热休克蛋白家族的研究进展[J].中国兽医科技,2004,34(5):33-37
[60]徐远胜.热休克蛋白20的心血管保护作用[J].国外医学·老年医学分册,2006,27(5):203-206
[61]Hase M,Depre C,Vatner SF,et al.H11 has dose-dependent and dual hypertrophic and proapoptotic functions in cardiac myocyte[J].Biochem J,2005,388(Pt2):475-483
[62]Natiol H,Powers SK,Demirel H A,et al.Exercise training increases heat shock protein in skeletal muscles of old rats[J].Med sci sports exercise,2001,33(5):729-734
[63]Samelman T R.Heat shock protein expression is increased in cardial and skeletal muscles of fischer 344 tats after endurance training[J].Expphysiol,2000,85(1):92-102
[64]Demirel H,Powers S,Cailland C,et al.Exercise training reduces myocardial lipid peroxidation following short-term ische mia referfusion[J].Med sci sports Exerc,1998,(30):1211
[65]Powers S K,Demirel H A,Vincent HK,et al.Exercise trainin improves myocardial tolerance to in vivo ische mia-reperfusion in the tat[J].A M.J physiol,1998(275):1468-1477
[66]Boguski M S,Mccormick,F.Proteins regulating ras and its relatives[J].Nature,1993:366-634
[67]陈磊.GEFs与小分子G蛋白的作用机制[J].医学理论与实践,2000,13(8):466-467
[1]常芸,林福美.运动性心肌肥大发生的调节因素[J].中国运动医学杂志,1995,14(1):21-26
[2]常芸.运动心脏重塑的调节[J].体育科学,2004,24(2):12-16
[3]Ostman I,Sjostrand NO,Swedin G.cardiac noradrenaline turnover and urinary catecholamine excretion in trained and untrained ratesduring rest and exercise[J].Acta Physioi Scand,1972,86(3):229-308
[4]张勇,刘健,李红艳,田振军.运动性心肌肥大的生物学机制[J].四川体育科学,2005,3(1):22-25
[5]Simpson P.stimulation of hypertrophy of cultured neonatal rat heart cells through an alpha 1-adrenergic receptor and induction of beating through analpha 1-and beta 1-adrenergic receptor interaction Evidence for independent regulation of growth and beating[J].Cir Res,1985,56(6):884-94
[6]Zanchetti A.et al.The renin-angiotensim system and heart,Am.J.Med,1985(85):3
[7]熊正英,等.运动性超负荷与压力超负荷大鼠心脏局部RAS系统变化的实验研究[J].体育科学,1996,16(4):61
[8]田振军.过度训练对大鼠心肌肾素-血管紧张素系统及有关酶的影响.体育科学,1999,19(5):67
[9]常芸,林福美,孙迎.运动心脏的自身调节因素[J].体育科学,1998,18(1):67-72
[10]李欣,潘珊珊.局部血管紧张素Ⅱ及其受体对运动心脏的影响[J].上海体育学院学报,2005,29(2):39-43
[11]李维根,高云秋,张继峰等.血管紧张素Ⅱ在运动性心肌肥大中的作用[J].中国运动医学杂志,1994,4(3):133-137
[12]田斌等.心脏肾素、血管紧张素系统在游泳引起的生理心肌肥大中的作用[J].生理学报,1994,46(5):509-513
[13]田振军,等.过度训练对大鼠心肌肾素-血管紧张素系统及酶的影响[J].体育科学,1999,19(5):67-70
[14]张镜如,乔健天.生理学[M].第4版.北京:人民卫生出版社,1997.387-393
[15]黄先玫,康曼丽.甲状腺素与心血管疾病[J].国外医学(心血管疾病分册),2000,27(4):199-201
[16]许玲,邓树勋.运动性心肌肥大的机理研究(综述)[J].体育科技,2001,22(1):37-41
[17]孙建波,路新国,张蕾,等.心钠素在医学与运动医学中的研究与进展[J].中国临床康复,2005,9(12):198-199
[18]郑澜,潘珊珊.运动与心钠素的研究现状与展望[J].成都体育学院学报,2001,27(4):92-96
[19]常芸,林福美,陈小同,罗安明.运动心脏重塑的发生与转归[J].体育科学,2000,20(5):46-50
[20]Maeda S,Miyuauehi Sakai S,et al.Prolonged exercise cause an increase in endothlin-1prodution in the heart in rats[J].A M J Physiol,1998,275:2105-2112
[21]常芸.运动心脏重塑的调节[J].体育科学,2004,24(2):12-16
[22]孙泊,庄涛.运动训练对大鼠心肌内皮素含量及受体分型的影响[J].中华物理医学与康复杂志,2006,2(11):743-746
[23]李昭波,高云秋,李维根,等.运动性和高血压性肥大心脏心肌内皮素及其基因表达的研究[J].中国运动医学杂志,2000,19(1):25-26
[24]马延超,常芸,张缨.运动性心脏重塑过程中内皮素基因的表达[J].中国运动医学杂志,2006,2(5):588-589
[25]常芸,孙迎,陈小同,等.运动心脏内分泌功能可复性的研究[J].中国运动医学杂志,1999,18(1):3-6
[26]Ie mitsu M,Miyauchi H,Maeda S,et al.Physiological and pathological cardiac hypertrophy induce different molecular phenotypes in the rat[J].Am J Physiol Regul Integr Comp Physiol,2001,281(6):2029-2036
[27]李红艳,田振军.运动性心肌肥大的生物学机制研究(综述)[J].西安体育学报,2004,21(5):53-55
[28]李昭波,高云秋.运动性和高血压性肥大心脏心肌内皮素及其基因表达的研究[J].中国运动医学杂志,2000,19(1):25-26
[29]赵海军,邓树勋.运动心脏及其形成机制(综述)[J].体育学刊,2002,9(2):121-123
[30]Ito H,HiroeM,HirataY,et al.Insulin like growth factor-1 induces hypertrophy with enhanced expression of muscle specific genes in cultured rat cardiomyocytes[J].Circulation,1993(5):1715-1721
[31]Cittadini A,Ishiguro Y,Stromer H,et al.Insulin-like growth factor 1 but not growth hormone augments mammalian myocardial contractility by sensitizing the myofilament to Ca~(2+)through a wortmannin-sensitive pathway:studies in rat and ferret isolated muscles [J].CirculRas,1998,8(3):50-59
[32]祁永梅,常芸.运动性心脏重塑过程中胰岛素样生长因子IGF-1基因的表达[J].中国运动医学杂志,2002,21(6):565-569
[33]常芸.运动心脏研究的现状与展望.山东体育科技,1999,21(4):28-34
[34]常芸,林福美,罗安明.细胞内游离钙在运动心脏重塑中的作用[J].中国运动医学杂志,1998,17(2):107-110
[35]Komupo I,Yazaki Y.Control of cardiac expression by mechanical stress[J].Annu RevPhysiol,1993,5(5):55-57
[36]Tanamura A.myocardial contractility and ventricular myosin isoenzymes is influented by cardiac hypertrophy and its regression[J].Basic Res Cardiol,1993,88(1):72-79
[37]常芸,赵中应,孙迎.耐力训练后大鼠心肌中原癌基因c-myc的表达[J].中国运动医学杂志,1999,18(2):111-114
[38]常芸,赵中应.耐力训练后大鼠心肌MLC-2基因的表达[J].中国运动医学杂志,2000,19(2):132-144