60%～70%最大摄氧量跑台运动对大鼠右心室肌蛋白质组差异表达的影响
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摘要
目的：本研究探讨4周60%～70%最大摄氧量跑台运动对大鼠右心室肌蛋白质组差异表达的影响,筛选出对运动应激敏感的目标蛋白质点,并对部分目标蛋白质的mRNA表达水平进行检测,为科学指导运动健身及慢性心血管疾病的运动康复研究提供新的思路及理论依据。
方法：将20只SD雄性大鼠随机分为对照组和实验组,以跑台运动的方式,复制递增运动负荷至24m-min-1的4周60%～70%最大摄氧量运动实验动物模型；提取右心室肌组织的全蛋白进行双向凝胶电泳分离,运用ImageMaster 2D Platinum图像分析软件对2-DE胶进行分析,选择差异表达上调5倍以上及下调至1/5以下的12个蛋白质点作为备选目标蛋白质点,用串联飞行时间质谱蛋白质仪(ULGRAFL-FLEX-TOF/TOF)进行质谱鉴定,以逆转录聚合酶链反应(RT-PCR)对部分目标蛋白质的mRNA表达水平进行检测。
结果：①大鼠经4周60%～70%最大摄氧量运动后,与对照组相比,心脏重量增加的百分率为0.62%,而心脏重量指数增加13.7%。心脏重量指数的增高有显著性差异(P<0.05)。②2-DE图谱上,对照组检测蛋白质点437±17个,实验组检测448±19个；运动后47个蛋白质点发生了较明显的变化。③通过对差异表达上调5倍以上及下调至1/5以下的12个点进行质谱鉴定,共鉴定出了11个蛋白,这些蛋白分别是：线粒体琥珀酸脱氢酶辅酶黄素蛋白亚基、线粒体-吡咯啉-5-羟酸酯脱氢酶、线粒体ATP合酶α亚基、Josephin-1、谷胱甘肽·S-转移酶Mu2、线粒体细胞色素b-cl联合体亚基、心肌肌动蛋白α、核苷二磷酸激酶B、微管动力调控蛋白、泛素融合降解1、腺苷酸激酶同工酶1。④逆转录聚合酶链实验结果显示：运动组大鼠心肌肌动蛋白α1、腺苷酸激酶同工酶1、谷胱甘肽S转移酶Mu2、核苷二磷酸激酶B、线粒体ATP合酶亚基a等的mRNA相对净光密度值低于对照组(p>0.05),表达水平下降；Josephin域蛋白1的mRNA相对净光密度值高于对照组(p>0.05),表达水平上升。
结论：①4周60%～70%最大摄氧量有氧运动后,大鼠心脏的形态学变化不明显。②筛选出11个与心肌的抗氧化能力、收缩能力、细胞分裂能力、能量代谢水平、物质代谢水平等密切相关的目标蛋白质。③目标蛋白质的差异表达一部分受其合成过程中上游基因转录水平的调控,一部分受下游翻译、修饰等的调控。
Objective:This paper is to explore the right ventricular proteomics'different expression under four weeks'60%～70% V02max intensity exercise stress, preliminarily screen out the target proteins that sensitive to exercise stress, and test the expression level of some target proteins'mRNA, these will provide new ideas and scientific theories for people's body-building and chronic cardio vascular disease' recovery.
Methods:Twenty SD male rats are randomly divided into the control group and the exercise group,then the exercise group are trained on treadmill by copying the 60%～70% V02max intensity exercise experimental animal models that workload increaing to 24 meters per minute,and then extract the whole proteins from the right ventricular, using the technology of 2DE to separate the proteins,applying the image analysis software (ImageMaster 2D Platinum) to analysis the 2DE gels,choosing the twelve protein spots that change more than 5 times between exercise for the first mass spectrum and the second mass spectrum,and adapting the method of RT-PCR to test the expression level of some target proteins'mRNA.
Results:①After four weeks'Aerobic training,compared to the control group,the weight of rats'heart increases by 0.62%,the weight index of rats'heart increases by 13.7%. the weight index of rats'heart increases significantly (P<0.05).②On the 2-DE map, the control group is detected 437±17 spots, the exercise group is detected 448±19 spots; there are 47 protein spots changing obviously③By mass spectrometry, spots that change more than 5 times between exercise are identified, these proteins are respectively:Succinate dehydrogenase [ubiquinone] flavoprotein subunit, Delta-1 -pyrroline-5-carboxylate hydrogenase of mitochondrial, ATP synthase subunit alpha of mitochondrial,Josephin domain-containing protein 1, Glutathione S-transferase Mu 2, Cytochrome b-cl complex subunit Rieske of mitochondrial, Actin of alpha cardiac muscle 1, Nucleoside diphosphate kinase B, Regulator of microtubule dynamics protein l,ubiquitin fusion degradation 1-like, Adenylate kinase isoenzyme 1.④Result of retrovirus polymerase chain experiment:In the mRNA expression level of alpha cardiac muscle 1,Adenylate kinase isoenzyme 1,Adenylate kinase isoenzyme 1, Glutathione S-transferase Mu 2, Nucleoside diphosphate kinase B, ATP synthase subunit alpha of mitochondrial,the exercise group is lower than the control group, expression level dropped.But the mRNA expression level of Josephin domain-containing protein 1, the exercise group is higher than than the control group, expression level ascended.
Conclusion:(1) By four weeks'Aerobic training, there is no change in morphological structure of rat's heart.(2) Eleven proteins are screened out,these proteins are closely related to the ability of cardiac muscle's shrinkage,oxidation·resistance,myocardial cell division, energy metabolism,material metabolism and and so on. (3) After four weeks'Aerobic training,one part of the target proteins's different expression is affected by the transcription of gene in proteins's synthesis process,and the other part of the target proteins's different expression is affected by the the translation、decoration of downstream.
方法：将20只SD雄性大鼠随机分为对照组和实验组,以跑台运动的方式,复制递增运动负荷至24m-min-1的4周60%～70%最大摄氧量运动实验动物模型；提取右心室肌组织的全蛋白进行双向凝胶电泳分离,运用ImageMaster 2D Platinum图像分析软件对2-DE胶进行分析,选择差异表达上调5倍以上及下调至1/5以下的12个蛋白质点作为备选目标蛋白质点,用串联飞行时间质谱蛋白质仪(ULGRAFL-FLEX-TOF/TOF)进行质谱鉴定,以逆转录聚合酶链反应(RT-PCR)对部分目标蛋白质的mRNA表达水平进行检测。
结果：①大鼠经4周60%～70%最大摄氧量运动后,与对照组相比,心脏重量增加的百分率为0.62%,而心脏重量指数增加13.7%。心脏重量指数的增高有显著性差异(P<0.05)。②2-DE图谱上,对照组检测蛋白质点437±17个,实验组检测448±19个；运动后47个蛋白质点发生了较明显的变化。③通过对差异表达上调5倍以上及下调至1/5以下的12个点进行质谱鉴定,共鉴定出了11个蛋白,这些蛋白分别是：线粒体琥珀酸脱氢酶辅酶黄素蛋白亚基、线粒体-吡咯啉-5-羟酸酯脱氢酶、线粒体ATP合酶α亚基、Josephin-1、谷胱甘肽·S-转移酶Mu2、线粒体细胞色素b-cl联合体亚基、心肌肌动蛋白α、核苷二磷酸激酶B、微管动力调控蛋白、泛素融合降解1、腺苷酸激酶同工酶1。④逆转录聚合酶链实验结果显示：运动组大鼠心肌肌动蛋白α1、腺苷酸激酶同工酶1、谷胱甘肽S转移酶Mu2、核苷二磷酸激酶B、线粒体ATP合酶亚基a等的mRNA相对净光密度值低于对照组(p>0.05),表达水平下降；Josephin域蛋白1的mRNA相对净光密度值高于对照组(p>0.05),表达水平上升。
结论：①4周60%～70%最大摄氧量有氧运动后,大鼠心脏的形态学变化不明显。②筛选出11个与心肌的抗氧化能力、收缩能力、细胞分裂能力、能量代谢水平、物质代谢水平等密切相关的目标蛋白质。③目标蛋白质的差异表达一部分受其合成过程中上游基因转录水平的调控,一部分受下游翻译、修饰等的调控。
Objective:This paper is to explore the right ventricular proteomics'different expression under four weeks'60%～70% V02max intensity exercise stress, preliminarily screen out the target proteins that sensitive to exercise stress, and test the expression level of some target proteins'mRNA, these will provide new ideas and scientific theories for people's body-building and chronic cardio vascular disease' recovery.
Methods:Twenty SD male rats are randomly divided into the control group and the exercise group,then the exercise group are trained on treadmill by copying the 60%～70% V02max intensity exercise experimental animal models that workload increaing to 24 meters per minute,and then extract the whole proteins from the right ventricular, using the technology of 2DE to separate the proteins,applying the image analysis software (ImageMaster 2D Platinum) to analysis the 2DE gels,choosing the twelve protein spots that change more than 5 times between exercise for the first mass spectrum and the second mass spectrum,and adapting the method of RT-PCR to test the expression level of some target proteins'mRNA.
Results:①After four weeks'Aerobic training,compared to the control group,the weight of rats'heart increases by 0.62%,the weight index of rats'heart increases by 13.7%. the weight index of rats'heart increases significantly (P<0.05).②On the 2-DE map, the control group is detected 437±17 spots, the exercise group is detected 448±19 spots; there are 47 protein spots changing obviously③By mass spectrometry, spots that change more than 5 times between exercise are identified, these proteins are respectively:Succinate dehydrogenase [ubiquinone] flavoprotein subunit, Delta-1 -pyrroline-5-carboxylate hydrogenase of mitochondrial, ATP synthase subunit alpha of mitochondrial,Josephin domain-containing protein 1, Glutathione S-transferase Mu 2, Cytochrome b-cl complex subunit Rieske of mitochondrial, Actin of alpha cardiac muscle 1, Nucleoside diphosphate kinase B, Regulator of microtubule dynamics protein l,ubiquitin fusion degradation 1-like, Adenylate kinase isoenzyme 1.④Result of retrovirus polymerase chain experiment:In the mRNA expression level of alpha cardiac muscle 1,Adenylate kinase isoenzyme 1,Adenylate kinase isoenzyme 1, Glutathione S-transferase Mu 2, Nucleoside diphosphate kinase B, ATP synthase subunit alpha of mitochondrial,the exercise group is lower than the control group, expression level dropped.But the mRNA expression level of Josephin domain-containing protein 1, the exercise group is higher than than the control group, expression level ascended.
Conclusion:(1) By four weeks'Aerobic training, there is no change in morphological structure of rat's heart.(2) Eleven proteins are screened out,these proteins are closely related to the ability of cardiac muscle's shrinkage,oxidation·resistance,myocardial cell division, energy metabolism,material metabolism and and so on. (3) After four weeks'Aerobic training,one part of the target proteins's different expression is affected by the transcription of gene in proteins's synthesis process,and the other part of the target proteins's different expression is affected by the the translation、decoration of downstream.
引文
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