【作者】 邵月；

【导师】 丁树哲；

【作者基本信息】 华东师范大学 ， 运动人体科学， 2010， 博士

【摘要】 细胞能量代谢异常和内外稳态环境紊乱是肿瘤和糖尿病发生的重要原因。骨骼肌是人体最大的运动和内分泌器官,骨骼肌细胞能量代谢相关基因表达对长期运动的适应是运动预防肿瘤发生、胰岛素抵抗和Ⅱ型糖尿病非常重要的因素。P53作为肿瘤抑制蛋白、细胞周期调控检查点、能量代谢的调节者、氧化应激的平衡者,位于多个细胞信号转导通路的核心位置,是生物体内部多个信号通路的调节者、平衡者、整合者,对调节细胞能量代谢、保持细胞氧化应激信号稳态和保持生物体内稳态效应都具有非常重要的作用,保持P53基因的稳态表达是预防肿瘤和延缓衰老的策略之一。体育锻炼能促进机体新陈代谢,延缓细胞衰老,减少细胞癌变几率,适宜的运动能够通过影响P53调节的能量代谢信号通路延续P53信号稳态。目的：本研究分别建立长期耐力训练模型、间歇性冲刺训练模型和一次急性运动模型,耐力训练模型以长时间、低强度为特征,间歇性冲刺训练模型以短时间、大强度、间歇性为特征,分别探讨、比较运动影响下SD大鼠、糖尿病GK大鼠骨骼肌的基因表达应答,期望能从基因表达水平上揭示骨骼肌细胞适应不同运动类型保持能量代谢和氧化应激平衡的分子机制,为运动保持骨骼肌正常生理功能和促进病理状态的改善和恢复提供一定的科学参考。方法：清洁级Sprague-Dawley雄性大鼠40只,约4周龄,体重100±5g,随机分为4组,即安静组(CON, n=10),耐力组(ET，n=10),冲刺组(SIT, n=10),急性组(AE,n=10)；糖尿病雄性大鼠12只,约8周龄,体重250±5g,随机分为2组,即GK安静组(GKC, n=6),GK耐力组(GKE, n=6)。耐力训练：正常SD大鼠和糖尿病GK大鼠均进行每天30-60min低强度(≤16.7m/min)的持续跑台运动；每周训练6天,训练6周。间歇性冲刺训练：正常SD大鼠每天9-10次10s最大强度(≥42m/min)的跑台运动,间歇时间30-60s,训练6周。最后一次训练结束后24h,所有大鼠依次断颈处死。一次急性运动：正常SD大鼠6周期间饲养环境等各方面均与安静组相同,进行一次60min低强度(≤16.7m/min)急性跑台运动后断颈处死。心脏取血,检测血清血糖、胰岛素、脂联素、甘油三酯、总胆固醇、糖化血清蛋白和红细胞糖化血红蛋白；取腓肠肌检测乳酸、GSH、GSSG含量；用实时荧光定量PCR法检测腓肠肌P53、SCO2、SCO1、COXⅡ、TIGAR、HKⅡ、PGM2、PDK4、PFKm、CPT1-β、AMPKa2、GLUT4的基因转录水平；用Western blot测定腓肠肌细胞P53、TIGAR、SCO2、SCO1、COXⅡ的蛋白表达水平。结果：(1)在正常生理条件下,一次急性运动、耐力训练和间歇性冲刺训练并没有从整体水平上影响机体血糖稳态和胰岛素抵抗指数,都显著降低了总胆固醇、甘油三酯和糖化血清蛋白水平,耐力训练还显著增加了脂联素分泌。在糖尿病病理条件下,耐力训练表现出良好的降低血糖、糖化血清蛋白、胰岛素抵抗指数、总胆固醇和升高脂联素的效果。(2)在正常生理条件下,一次急性运动、耐力训练、间歇性冲刺训练对P53基因转录和蛋白表达均没有产生显著性影响。在病理条件下,耐力训练对P53基因表达的影响与正常生理状态下不同,P53基因转录和蛋白表达均显著降低。(3)在正常生理条件下,一次急性运动显著增加了SCO2基因转录,对SCO2蛋白表达、SCO1和COXⅡ基因表达均没有产生影响。耐力训练显著增加了SCO2、SCO1基因转录,对COXⅡ基因转录影响不大,但却显著增加了SCO2、COXⅡ蛋白表达水平。间歇性冲刺训练对SCO2基因转录没有影响,非常显著增加了SCO1、COXⅡ基因转录水平,同时显著增加了SCO2和COXⅡ的蛋白表达水平。三种运动方式都显著升高了GSH/GSSG比率。在病理条件下,耐力训练没有影响SCO2、SCO1基因表达,而且还极大降低了COXⅡ蛋白表达水平。但GSH/GSSG比率却显著上调。(4)在正常生理条件下,一次急性运动显著增加了TIGAR、PGM2基因转录,对TIGAR蛋白表达、HKⅡ和PFKm基因转录、乳酸含量均没有产生影响。耐力训练显著增加了TIGAR基因转录和蛋白表达水平,但对PGM2、HKⅡ、PFKm和乳酸含量均没有产生影响。间歇性冲刺训练非常显著增加了PFKm基因转录和乳酸含量,但对TIGAR基因表达、PGM2、HKⅡ、PFKm基因转录没有产生影响。在病理条件下,耐力训练显著增加了HKⅡ、PFKm、GLUT4、AMPKα2基因转录,但对TIGAR基因表达、PGM2基因转录和乳酸含量没有影响。(5)在正常生理条件下,一次急性运动非常显著降低了PDK4基因转录水平,对CPT-1β则没有影响。耐力训练在非常显著降低PDK4基因转录水平的同时非常显著升高了CPT-1β基因转录水平。间歇性冲刺训练使PDK4、CPT-1β基因转录水平均显著升高。在糖尿病病理条件下,耐力训练显著降低了GK大鼠PDK4水平,但是,耐力训练并没有像在正常生理条件下一样对大鼠CPT-1β基因表达产生影响。结论：(1)在正常生理条件下,从血液指标来看,运动并没有从整体水平上影响机体的正常生理稳态,机体的整体生理调节尚处于稳态范围内,运动对改善健康机体整体水平糖脂代谢能力具有一定效果。在糖尿病病理条件下,从整体水平上来讲,耐力训练可能是改善糖尿病高血糖症状的有效方式。(1)在正常生理条件下,运动对P53没有产生显著性影响,而是促进P53充分发挥其调节、检查、平衡、整合的稳态效应以保持机体发挥正常生理功能。在病理条件下,耐力训练使P53基因表达能力下降,可能是受糖尿病病程的影响,P53无法继续保持机体生理功能在正常稳定状态,其基因表达能力的下降极有可能是为了促进细胞的生存,也许是为了延缓GK大鼠病态细胞的凋亡和衰老进程。(2)在正常生理条件下,从SCO2、COXⅡ基因表达结果可以看出,耐力训练和间歇性冲刺训练对长期训练能产生很好的运动适应,都呈现出促进线粒体有氧呼吸效应,一次急性运动则没有使有氧呼吸链组分改善达到期望效果。SCO1基因表达似乎对运动方式不敏感,其基因转录水平的极显著性升高不能排除最后一次训练短时的应激反应。从对GSH/GSSG比率的影响来看,运动可能都改善了机体的氧化还原环境。在糖尿病病理条件下,似乎耐力运动对线粒体有氧呼吸轴并没有产生积极而有效的影响,综合P53和GSH/GSSG结果来看,也许是P53促生存功能所致。在病理条件下,也许细胞更重要的功能不是提高运动能力,而是积蓄一切力量,促使细胞生存。(3)在正常生理条件下,耐力训练诱导的TIGAR基因表达水平显著上调必将有利于机体能量代谢转向更经济高效的有氧呼吸通路,是机体对耐力训练产生的一种良好的运动适应现象。运动没有对骨骼肌摄取糖的能力造成大的冲击,这种对血糖稳态的有力控制对保持机体正常生理功能的发挥具有重要的意义。运动诱导的P53基因的稳态表达似乎也保持了糖酵解通路其直接靶基因的稳态表达,耐力训练对线粒体有氧呼吸通路具有较好的促进作用,但对糖酵解通路影响不是太大。冲刺训练诱导的乳酸产生与PFKm基因转录水平呈现一致性升高,可在一定程度上印证间歇性冲刺训练能量产生很可能以无氧代谢为主,这与其运动方式能量代谢需求特点相吻合。耐力训练降低了糖尿病大鼠的氧化应激能力,这也许是耐力训练对糖尿病大鼠细胞生存能力的一大贡献。(5)在糖尿病病理条件下,耐力训练极有可能通过能量敏感性通路AMPK-GLUT4葡萄糖转运机制极大地促进了骨骼肌糖摄取能力,对于改善GK大鼠高血糖症状是一种非常有效的方式。但结合P53基因表达水平显著下调结果来看,GLUT4和HKⅡ好像失去了P53对其直接抑制作用,朝向促进糖酵解的方向发展,下调的P53亦不能通过TIGAR来抑制糖酵解通路。这些变化虽然降低了血糖,但也有可能为恶性病变埋下隐患,可能是机体对运动并没有很好适应的一种表现。也许在病理状态下,细胞分子之间的信号转导通路可能会发生一定的改变,本实验耐力训练方式未必对糖尿病大鼠完全有利,运动对糖尿病的重要意义也许重在防而不是治。(6)在正常生理条件下,耐力训练非常显著减弱了PDK4对PDC的磷酸化抑制,加强了骨骼肌线粒体丙酮酸氧化能力,同时非常显著增加了CPT-1β表达,也加强了脂肪酸氧化能力,表明耐力训练是激活线粒体呼吸的有效运动方式。间歇性冲刺训练极有可能加强了PDK4对PDC的磷酸化抑制,其供能可能以糖酵解为主,但其同时也升高了CPT-1β表达,表明间歇性冲刺训练也有可能是激活线粒体呼吸的一种有效运动方式。在糖尿病病理条件下,耐力训练显著性降低了GK大鼠PDK4水平,对促进糖尿病大鼠有氧呼吸能力是有利的,也是运动能改善糖尿病症状的一种分子水平上的依据,但是,耐力训练并没有像在正常生理条件下一样对大鼠CPT-1β基因表达产生良好的适应性反应。(7)综合结果显示,在正常生理条件下,不同的训练方式对机体不同组分产生的影响也各不相同,长期运动训练更能使机体产生较好的运动适应。冲刺训练与耐力训练相比,在诱导肌肉运动适应方面具有相似性,可能是一种更有效的时间节省化方式。(8)在运动影响下的P53调节能量代谢通路中,并不是每一个因子都遵循其能量产生需求方式,尤其在糖尿病病理状态下,耐力训练糖尿病大鼠各因子变化有些甚至是明显的无氧供能特征。可能在病理状态下,运动确实影响了能量代谢途径,有些有利于机体的恢复与健康,而有些指标的改变却对机体产生了不良的影响。运动对机体的影响非常复杂,我们不能仅从一个方面或很少的几个方面来简单地下结论,运动对机体是有益还是有害,而是应该综合地观察其效果,毕竟整体水平机能的改善才是我们追求的终极目标。更多还原

【Abstract】 Cellular energy metabolism abnormality and the steady-state disorder of internal and external environment are the important reasons leading to cancer and diabetes. As the body’s largest sports and endocrine organs,the adaptation of genes-related expression of energy metabolism signal pathway to long-term exercise in skeletal muscle is considered to a very important factor for preventing the development of tumor, insulin resistance and type II diabetes.As a tumor suppressor,cell-cycle checkpoint,the regulator of energy metabolism,and the balancer of oxidative stress, P53 lies in the center of many Cellular signaling pathway,and is their regulator,balanc-er,and integrator.Therefore,P53 has a very important role in regulating cellular energy metabolism,keeping oxidative stress balance and the steady-state of organism.One of the strategies is to maintain the gene expression steady-state of P53 for preventing cancer and premature aging.exercise can promote the body’s metabolism,delay cellular senescence,reduce chances of cancerous cells,therefore,appropriate exercise may continue the P53 signal steady-state by regulating the signaling pathway of energy metabolism.Purpose:Training program of endurance training and sprint interval training and an acute endurance training were founded in the study,Endurance training model is based on a long,low-intensity and Sprint interval training model is based on short-term, high-intensity.one of the purposes is to determine the gene expression to training program in physiological and pathological conditions,expected to reveal the molecular mechanism of skeletal muscle cell’s adaption to training program, and to explore the cause of keeping the balance of energy metabolism and oxidative stress from the level of gene expression,and to provide some evidences that exercise can improve the physiological function of skeletal muscle and promote the improvement and recovery of DM.Methods:40 male Sprague-Dawley rats were distributed into four groups:sedentary(CON,n=10),an acute endurance training(AE,n=10),endurance training(ET,n=10),sprint interval training(SIT,n=10).Endurance training consisted of 30-60min of continuous threadmill exercise at a lower intensity(<16.7m/min)per day,6 days/wk.Sprint interval training consisted of 9-10 repeats of a 10s"all ouf"threadmill test(≥42m/min)with 30-60s recovery between repeats,6 days/wk. After 6 weeks of either sprint interval or endurance training,the rats of group CON,ET and SIT were decapitated 24h after the last threadmill test.Group AE were administered similarly to Group CON in the 1-6wk,After an acute endurance training of 60min of continuous threadmill exercise at a lower intensity(<16.7m/min),the rats of Group AE were decapitated too.Blood was collected from heart,blood glucose,insulin, adiponectin,triglyceride,total cholesterol,glycated serum protein and glycolated hemoglobin were detected.The content of lactate,GSH and GSSG in gastrocnemius homogenate were measured by spectrophotometric assays.Real-time PCR was used to determine the mRNA content of P53,SCO2,SCO1,COXⅡ,TIGAR,HKII,PGM2, PDK4,PFKm,CPT1-β,AMPKa2,GLUT4 in gastrocnemius.Western blot was used to determine the protein content of P53,TIGAR,SCO2,SCO1,COXⅡin gastrocnemius.Results:(1) In the normal physiological conditions,AE,ET and SIT did not affect the blood glucose homeostasis and insulin resistance index from the overall level,but significantly downregulated the levels of total cholesterol,triglyceride,and glycosylated serum protein,ET also significantly increased adiponectin secretion.In the pathological conditions of diabetes,ET showd the sound effect in increasing adiponectin and reducing blood glucose,glycosylated serum protein,insulin resistance index and total cholesterol.(2) In the normal physiological conditions,AE,ET and SIT have no significant impact in both gene transcription and protein expression of P53.However,it Shows a different result in the pathological conditions,P53 gene transcription and protein expression were all significantly reduced.(3) In the normal physiological conditions,AE significantly increased SCO2 gene transcription,but had no effect in its protein expression,and had no effect in gene expression of SCO1 and COXⅡ.ET obviously upregulated the gene expression of SCO2 and SCOl,but had no effect in COXⅡ,however,ET clearly increased the protein expression of SCO2 and COXII.SIT had no effect in SCO2 gene transcription,but very significantly increased the gene transcription of SCO1 and COXII,in addition,SIT obviously upregulated the protein expression of SCO2 and COXⅡ.All AE,ET and SIT clearly elevated the ratio of GSH/GSSGIn the pathological conditions,ET have no effect in gene expression of SCO2 and SCOl,and significantly reduced the COXII protein expression,but the ratio of GSH/GSSG clearly elevated.(4)In the normal physiological conditions,AE significantly increased the gene transcription of TIGAR,PGM2,but had no effect in TIGAR protein expression and the content of lactate and the gene transcription of HKII and PFKm.ET clearly upregulated TIGAR gene expression from both gene transcription and protein expression,but had no effect in the content of lactate and the gene transcription of PGM2,HKII and PFKm.SIT very significantly increased the gene transcription of PFKm and the content of lactate,but had no effects in TIGAR gene expression and the gene transcription of PGM2,HKII and PFKm.In the pathological conditions,ET significantly increased the gene transcription of HKII,PFKm,GLUT4 and AMPKa2,but had no effect in gene expression of TIGAR,gene transcription of PGM2 and the content of lactate.(5) In the normal physiological conditions,AE very significantly reduced the gene transcription of PDK4,and had no effect in CPT-1β.ET very significantly reduced the gene transcription of PDK4 but very significantly increased the leval of CPT-1β.SIT clearly upregulated the gene transcription of both PDK4 and CPT-1β.In the pathological conditions,ET obviously downregulated the leval of PDK4,but had no effect in CPT-1β.Conclusions:(1) In the normal physiological conditions,in terms of the blood indicators,Exercise did not affect the body’s normal physiological steady-state from the overall leval,and has a certain effect in improving the glucose and lipid metabolism.In the pathological conditions,ET may be an effective way to improve the high blood sugar symptoms of diabetes.(2) In the normal physiological conditions,exercise has no obvious effect in P53, its important role might be to promote the functions of regulation, inspection, balance, integration of P53,all of these were to maintain the normal physiological function.In the pathological conditions,ET downregulated the gene expression of P53.Might be affected by DM,the body’s normal steady-state couldn’t be maintained by P53,the downregulation might be to promote cell survival and delay the cell’s apoptosis and aging of GK rats.(3) In the normal physiological conditions,the improvement of mitochondrial aerobic respiration induced by ET and SIT might be a good adaption to long-term training,but AE did the opposite.SCO1 gene expression seemed insensitive to training programe,the significant upregulation of gene transcription level might be the short-term stress response of the last training.As to the rise of GSH/GSSG under three exercise modes,exercise might have improved the environment of the body’s redox.In the pathological conditions,ET seemed have no effective influence in mitochondrial aerobic respiration,speculating from the comprehensive results,its important role might be to promote the cell’s survival.Therefore,we could make a conclusion that the important function of P53 is riot to enhance exercise ability but to save all forces to promote cell survival under the pathological conditions.(4) In the normal physiological conditions,the significant upregulation of TIGAR gene expression induced by ET Will help the body’s energy metabolism shift to the more cost-effective aerobic respiration pathway,and is a good adaptation to ET. Training hadn’t cause the adverse effects to the uptaking of glucose,the effective control of blood glucose homeostasis will have an important significance in maintaining normal physiological function of the body.The P53 steady-state induced by training seems keep the steady-state of P53 target gene in the glycolytic pathway, ET has a role in promoting mitochondrial respiration but has little impact on the glycolytic pathway.Lactic acid generation and PFKm gene transcription shows obvious increase,To some extent,the energy production induced by SIT is mainly likely to anaerobic metabolism,and this is consistent with its energy metabolism characteristics.ET downregulated the GK rats’ ability of oxidative stress,this may be an contribution to diabetic rat cells’survival.(5) In the pathological conditions,ET may be significantly contribute to the glucose uptake ability of skeletal muscle through AMPK-GLUT4 glucose transport mechanism that is the energy-sensitive pathway,and this is a effective way to improve the high blood sugar.But considered the obvious downregulation of P53 gene expression,GLUT4 and HKII seem to have lost the direct inhibition by P53,and developed towards glycolysis,P53 also can not suppress glycolysis pathway by TIGAR.Although these changes reduced the blood sugar,it still may be induce the malignant lesions,it shows that the body is not well adapted to the training.the transduction pathway may occur some changes under the pathological conditions,in this study,ET may not be entirely beneficial to DM rats,the important significance of exercise to diabetes may be lies in prevention rather than treatment.(6) In the normal physiological conditions,ET significantly diminished the phosphorylation of PDK4 on the PDC,strengthened the pyruvate oxidation of skeletal muscle.At the same time,CPT-1βexpression very significantly increased,also strengthened the capacity of fatty acid oxidation.All these shows that ET is an effective way to activate mitochondrial respiratory. SIT may strengthen the phosphorylation of PDK4 on the PDC,which main energy supply is likely to glycolysis,but simultaneously increased the CPT-1βexpression.All these shows that SIT might be an effective way to activate mitochondrial respiratory too.In the pathological conditions,ET obviously reduced the PDK4 leval of GK rats,it was beneficial to GK rat’s aerobic respiration,and is also a molecular basis that exercise can improve symptoms of diabetes.however,the CPT-1βgene expression hasn’t occurred the same good adaptation to ET as the same as in the normal physiological conditions.(7)A11 comprehensive results shows that different training programe has the different effect,long-term exercise enables the body to produce the better adaptation. Compared with ET and SIT,both has a similar adaptation,thus,SIT may be a more effective way of saving time.(8)When organism is in training,not every factor follows its own necessary energy generation way in energy metabolism pathway regulated by P53,Especially in the pathological conditions of DM,some changes of GK rats induced by ET show the obvious characteristics of anaerobic energy supply,exercise does affected the energy metabolism pathway,some may be beneficial to body’s retrieval and healthy,but others may be adverse.The impact induced by exercise is very complex,we couldn’t make the simple conclusion that exercise is beneficial or harmful to the body from a few aspects but should consider from the comprehensive aspects.After all,the improvement of function from the overall leval is our ultimate goal.更多还原