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间歇运动和rhG-CSF激活FGF1-PI3K-AKT通路促进心肌细胞增殖改善心梗大鼠心功能
【作者】 史秀超；
【导师】 田振军；
【作者基本信息】 陕西师范大学 ， 运动生物学， 2017， 博士
【摘要】 背景与目的:心肌梗死(myocardial infarction,MI)引起局部心肌缺血缺氧,导致大量心肌细胞死亡,发生心肌替代性纤维化,进而导致心功能恶化,诱发心衰。外源性重组粒细胞集落刺激因子(rhG-CSF)干预可动员骨髓干细胞(BMCs)入血,归巢至心梗部位,分化为有功能的心肌细胞,修复受损心脏。间歇运动是心脏康复的有效手段,但其作用机制不清。间歇运动是否通过动员内源性BMCs进入循环并发生归巢,促进心肌梗死心脏的心肌细胞再生,改善心功能,尚缺乏文献报道。FGF1-PI3K-AKT通路在细胞增殖中发挥重要作用。但关于FGF1在间歇运动和(或)rhG-CSF激活FGF1-PI3K-AKT通路,促进心肌细胞增殖并改善心梗大鼠心功能中能否发挥作用,尚缺乏实验证据。本文采用大鼠心梗模型,通过间歇运动和(或)rhG-CSF干预,以及骨髓干细胞受体阻断剂AMD3100或外源性注射重组大鼠FGF1干预,对心梗心脏形态结构与功能、心肌细胞钙瞬变与舒缩功能、心肌细胞增殖与凋亡及相关信号分子进行实验研究,将对心梗心脏疾病的防治手段与方法筛选提供理论和实验依据。其目的是探讨间歇运动和外源性rhG-CSF干预对心梗大鼠心肌细胞增殖的影响及可能机制。研究方法:在第二部分和第三部分中,雄性SD大鼠,随机分为假手术组(Sham组)、心梗组(MI组)、心梗+间歇运动组(ME组)、心梗+粒细胞集落刺激因子组(MG组)、心梗+粒细胞集落刺激因子+间歇运动组(MGE组)、心梗+AMD3100组(MA组)和心梗+ AMD3100+间歇运动组(MAE组),共7组,手术成功后每组12只。采用左冠脉前降支(LAD)结扎法建立心梗模型。第四部分有两种实验模型:第一种,雄性SD大鼠,假心梗+溶剂组(SV组)、假心梗+重组大鼠FGF1组(SF组)、心梗+溶剂组(MV组)和心梗+重组大鼠FGF1组(MF组),共4组,手术成功后每组12只;第二种,雄性SD大鼠,随机分为Sham、MI、ME、MG和MGE,共5组,手术成功后每组12只。G-CSF、AMD3100和重组大鼠FGF1给药方案:MG组和MGE组手术lh后皮下注射rhG-CSF,lOμg/kg/d×5d。MA组和MAE组术后lh腹腔注射AMD3100,20μg/kg/d×5d。Sham、MI组和ME组注射等量生理盐水对照。SF组和MF组术后第3d尾静脉注射重组大鼠FGF1(0.05mg/ml),2ml/kg/d×7d。SV组和MV组假手术后第3d尾静脉注射溶剂对照(0.01M pH7.4 PBS,肝素钠50U/ml,0.1%BSA),2ml/kg/d×7d。间歇运动方案:术后第8d开始进行为期3wk跑台间歇运动。每天开始时以10m/min×lOmin 热身运动;之后采用 25m/min×8min(85-90%of V02max)和15m/min×2min(50-60%of VO2max)交替进行 4 个循环训练;最后以 25m/min×8min和 10m/min×2min 结束训练,1h/d,5d/wk×3wk。干细胞动员效果测试:使用“动物外周血和脏器组织单核细胞分离试剂盒操作说明”分离外周血单个核细胞。抗体标记,流式细胞仪检测c-kit+、CD29+和CD34+细胞百分比。心功能、心肌细胞钙瞬变与收缩能力、心肌细胞增殖测试:彩色多普勒超声诊断仪测定左室射血分数和左室短轴缩短率;PowerLab/8S生理记录仪测定血流动力学指标;IonOptix测定急性分离的单个心肌细胞钙瞬变和收缩功能;TTC染色测定心肌梗死面积;PCNA和cTnT免疫荧光法检测新生心肌细胞百分率,激光共聚焦显微镜观察心肌细胞增殖情况。心肌细胞增殖相关信号分子检测:运用反转录PCR法检测基因表达。Western Blot 法检测 HIF-1、SDF-1、c-kit、CD29、CD34、Bcl-2、BAX、Caspase9、FGF1、FGF1R、PI3K、P-PI3K、AKT、P-AKT、mTOR、p-mTOR、P70 S6K、P-P70 S6K、cyclinBl、cyclinDl 蛋白表达。研究结果:1间歇运动和(或)rhG-CSF可有效刺激心梗大鼠BMCs动员与归巢(1)心肌梗死和rhG-CSF均可显著增加外周血c-kit+、CD29+和CD34+细胞数量,CXCR4受体阻断剂AMD3100可减弱其效应;间歇运动可显著增加心肌梗死大鼠的外周血c-kit+、CD29+和CD34+细胞数量,间歇运动和rhG-CSF共同作用效应更显著。表明,间歇运动和(或)rhG-CSF干预可显著动员骨髓干细胞进入循环系统。(2)心肌梗死和rhG-CSF均可显著增加心梗边缘区心肌组织HIF-lα和SDF-1的表达,CXCR4受体阻断剂AMD3100可减弱其表达;间歇运动可显著增加心梗边缘区心肌组织HIF-lα和SDF-1的表达,间歇运动和动员剂rhG-CSF共同作用效应更显著。表明,间歇运动和(或)rhG-CSF干预可显著增加干细胞趋化动力。(3)心肌梗死和rhG-CSF均可显著增加心梗边缘区心肌组织c-kit、CD29和CD34的表达,CXCR4受体阻断剂AMD3100可减弱其表达。间歇运动可显著增加心梗边缘区心肌组织c-kit、CD29和CD34的表达,间歇运动和rhG-CSF共同作用效应更显著。表明,间歇运动和(或)rhG-CSF干预可显著增加干细胞的归巢效应。2间歇运动和(或)rhG-CSF显著刺激心梗大鼠心肌细胞再生改善心功能(4)间歇运动或rhG-CSF均可显著增加心梗大鼠心肌细胞PCNA+cTnT+双阳性细胞数量,该效应可被CXCR4受体阻断剂AMD3100显著减弱。间歇运动和rhG-CSF共同作用效果更加显著。(5)间歇运动干预显著缩小梗死面积百分比,该作用可被CXCR4受体阻断剂AMD3100显著减弱。间歇运动和rhG-CSF共同干预效应尤其明显。(6)间歇运动干预后,心梗边缘区钙瞬变和舒缩参数单个心肌细胞Ca2+荧光强度(340/380nm)比值和[Ca2+]i变化幅度、Ca2+荧光达基线50%时间、心肌细胞收缩最大速率均显著增加,Ca2+荧光达峰值50%时间和心肌细胞舒张最大速率均显著降低;超声心动参数LVEF及LVFS、血流动力学参数LVSP、LV+dp/dtmax和LV-dp/dtmax显著升高,LVEDP显著下降,表明间歇运动干预后心梗边缘区单个心肌细胞收缩和舒张功能以及整体心脏功能均大幅提升。该作用可被CXCR4受体阻断剂AMD3100显著减弱。间歇运动和rhG-CSF共同干预效应更显著。3 FGF1在间歇运动和(或)rhG-CSF激活FGF1-PI3K-AKT通路,促进心肌细胞增殖并改善心梗大鼠心功能中发挥重要作用(7)外源性重组大鼠FGF1显著升高心梗大鼠循环FGF1浓度和心梗心肌组织的FGF1含量,显著增加PCNA+cTnT+双阳性细胞数量。显著降低心梗大鼠心肌组织caspase 3的活性。(8)间歇运动和(或)rhG-CSF共同作用均显著降低心梗大鼠梗死边缘区p-caspase9和BAX表达,显著升高Bcl-2和Bcl-2/BAX比值。表明,间歇运动或rhG-CSF均显著降低心肌细胞凋亡,间歇运动和rhG-CSF共同作用效果更显著。(9)间歇运动和(或)rhG-CSF共同作用均显著升高心梗心脏FGF1、FGF1R、mTOR、p-AKT、p-PI3K P85、p-P70S6K、cyclin B1 和 Cyclin D1 蛋白和NKX2.5和GATA4mRNA表达水平。表明,心梗心肌FGF1-PI3K-AKT通路和心肌细胞增殖发生代偿性激活。间歇运动显著激活心梗心肌FGF1-PI3K-AKT通路,促进心肌细胞增殖;间歇运动和rhG-CSF共同作用效果更加显著。(10)间歇运动促进NKX2-5和GATA4 mRNA表达,促进梗死心脏再生、降低心肌细胞凋亡、激活FGF1-PI3K-AKT通路、促进心肌细胞增殖的效应,与rhG-CSF干预的效果相同。研究结论:1间歇运动和(或)rhG-CSF干预,可有效促进心梗大鼠骨髓干细胞动员进入循环,在趋化因子SDF-1作用下归巢至心肌梗死区及梗死边缘区;间歇运动和rhG-CSF共同作用的效应优于单一干预因素。2间歇运动和(或)rhG-CSF干预,可有效促进心肌细胞增殖,降低心肌细胞凋亡,改善心肌细胞钙瞬变和收缩功能,整体提升心梗大鼠心功能。3外源性注射重组大鼠FGF1显著升高心梗大鼠循环和心肌组织FGF1水平,显著促进心肌细胞增殖,抑制心肌细胞凋亡。4心梗大鼠心肌FGF1-PI3K-AKT通路和心肌细胞增殖被代偿激活;间歇运动显著激活心梗大鼠心肌FGF1-PI3K-AKT通路,显著增加心肌细胞增殖的数量;间歇运动和rhG-CSF共同作用效果更加显著。表明,FGF1在间歇运动和(或)rhG-CSF激活FGF1-PI3K-AKT通路,促进心肌细胞增殖并改善心梗大鼠心功能中发挥重要作用。
【Abstract】 Background:Myocardial infarction(MI)causes hypoxia in myocardium,leads to numbers of cardiomyocytes apoptosis or necrosis and cardiac fibrosis,which would induce heart dysfunction and heart failure(HF).Recombined human granulocyte cell-stimulating factor(rhG-CSF)is an effective bone marrow cell(BMCs)mobilization reagent.BMCs could be mobilized into blood circulation by rhG-CSF,and home to infarction region,differentiate into functional cardiomyocytes to repair the injured heart.Interval exercise is an effective method of cardiac rehabilitation,but its mechanism is still not well known.It has no report about whether interval exercise can mobilize endogenous BMCs into blood and induce them homing to the infarcted heart,improve cardiac regeneration.Fibroblast growth factor 1(FGF1)-PI3K-AKT plays a very important role in cell proliferation.However,whether interval exercise with or without rhG-CSF treatment could activate theFGF1-PI3K-AKT signaling and promote cardiomyocyte proliferation after MI are still not clear.In this study,rats were used to establish the MI model and subjected to interval exercise with or without rhG-CSF injection,or CXCR4 antagonist AMD3100 or exogenous recombinant rat FGF1 injection.The heart function,cardiac morphology,cardiomyocyte calcium transients,proliferation and apoptosis,and related molecule mechanism were detected.The aim of this study was to study the effect and possible mechanism of interval exercise with or without rhG-CSF treatment on improving cardiomyocyte proliferation in rats with MI.Methods:3-month old Sprague-Dawley rats were used to establish MI model by ligation of the left anterior descending coronary artery,and randomly divided into 7 groups in part 2 and part3:Sham group(Sham),MI group(MI),MI with interval exercise group(ME),MI with rhG-CSF treatment group(MG),MI with rhG-CSF treatment and interval exercise group(MGE),MI with AMD3100 group(MA)and MI with AMD3100 and interval exercise group(MAE),12 rats in each group.In part 4,we have two experimental model:First,rats were divided into Sham with solvent group(SV),Sham with recombinant rat FGF1 group(SF),MI with solvent group(MV)and MI with recombinant rat FGF1 group(MF);The other,rats were divided into Sham,MI,ME,MG and MGE,12 rats in each group.The rats in MG and MGE groups were injected with rhG-CSF(physiological saline solution diluted)by subcutaneous injection,10 μg/kg/d×5d.The rats in MA and MAE groups were injected with AMD3100 intraperitoneally,200 μg/kg/d×5d.The rats in Sham,MI and ME groups were injected the same volume of physiological saline solution as control.The rats in SF and MF groups were administered with recombinant rat FGF1,2ml/kg/d×7d,and the rats in SV and MV groups were injected with solvent as control.Rats in ME,MGE and MAE groups were subjected to interval exercise.The training started at the 8th day after surgery.First,rats were warmed up at 10m/min×10min on the treadmill.Then,25m/min×8min and 15m/min×2min.Repeat it for four times.Finally,25m/min×8min and 10m/min×2min and terminated the training.1h/d,5d/wk×3wk.Peripheral blood mononuclear cells were isolated by reagent kit.Flow cytometry were used to detect the level of c-kit+,CD29+ and CD34+ cells;Cardiac function was detected by color doppler ultrasonic diagnostic instrument and hemodynamic measurement;Individual cardiomyocytes were isolated and the calcium transients and systolic function were detected by single-celled detection system(IonOptix);TTC staining were used to determine the infarcted area;PCNA+ and cTnT+ cardiomyocytes were counted by immunofluorescence staining to analyze cardiomyocyte proliferation;Cardiac transcription factors GATA4 and NKX2-5 gene expression was measured by reverse transcription polymerase chain reaction(RT-PCR),and HIF-1,SDF-1,c-kit,CD29,CD34,Bcl-2,BAX,Caspase 9,FGF1,PI3K,P-PI3K,AKT,P-AKT,P70 S6K,P-P70 S6K,cyclin B1,and cyclinD1 were detected by Western Blot.Results:1.Interval exercise with or without rhG-CSF treatment could stimulate BMCs mobilization in MI rats.(1)MI and rhG-CSF could enhance the level of c-kit+,CD29+ and CD34+mononuclear cells in peripheral blood,which could be reduced by AMD3100.Interval exercise after MI could increase the number of c-kit+,CD29+ and CD34+ mononuclear cells in peripheral blood,and combined with rhG-CSF gave a better effect.These results indicated that interval exercise with or without rhG-CSF treatment can significantly mobilize BMCs into the circulation system.(2)MI and rhG-CSF could up-regulate the expression of HIF-la and SDF-1 in the peri-infarcted area of rats,which could be reduced by AMD3100.Interval exercise after MI could increase the expression of HIF-1α and SDF-1,and combined with rhG-CSF gave a better effect.These results indicated that interval exercise with or without rhG-CSF treatment could improve the chemotaxis capability of BMSCs after MI.(3)MI and rhG-CSF could up-regulate the protein expression of c-kit,CD34 and CD29 in the peri-infarcted area of rats,which could be inhibit by AMD3100.Interval exercise after MI could increase the expression of c-kit,CD34 and CD29,and combined with rhG-CSF gave a better effect.These results indicated that interval exercise with or without rhG-CSF treatment could stimulate BMSCs homing to infarted heart.2.Interval exercise with or without rhG-CSF treatment could stimulate cardiomyocyte proliferation and improve cardiac function in MI rats.(4)Interval exercise and rhG-CSF treatment could increase PCNA+cTnT+ cells significantly in MI rats.The effect was weakened by AMD3100.Combined use of interval exercise and rhG-CSF had a better effect.(5)Interval exercise could reduce infarcted size in MI rats.The effect was weakened by AMD3100.Combined use of interval exercise and rhG-CSF had a better effect.(6)Interval exercise could increase the amplitude of calcium transient and intracellular Ca2+ concentration,decrease the time from peak value to 50%baseline and the time from the baseline to 50%peak;the velocity of monocardiomyocyte contracting increased in ME and MGE group rats significantly contrast to that of MI group rats.It also increased the systolic and diastolic function of cardiomyocytes in the peri-infarct region in MI rats.The effect was weakened by AMD3100.Combined use of interval exercise and rhG-CSF had a better effect.3.Interval exercise with or without rhG-CSF treatment could activateFGFl-PI3K-AKT signaling pathway,improve cardiomyocyte prolifer-ation and cardiac function in MI rats.(7)rhG-CSF could increase the level of FGF1 in peripheral blood and myocardium,and increase the number of PCNA+ cTnT+ cells,reduce the activation of caspase3 in the peri-infarcted area.(8)Interval exercise with or without rhG-CSF treatment could down-regulate the protein expression of p-caspase 9 and BAX,up-regulate the Bcl-2 expression and the ratio of Bcl-2/BAX,reduced cell apoptosis.(9)Interval exercise with or without rhG-CSF treatment could up-regulate the protein expression of FGF1,FGFR,mTOR,p-AKT,p-PI3K P85,p-P70S6K,cyclin B1 and Cyclin D1 and the mRNA expression of GATA4 and NKX2-5 in the peti-infarcted area of MI rats,and combined use of interval exercise and rhG-CSF had a better effect.Interval exercise could activate theFGF1-PI3K-AKT signal pathway,improve cardiomyocyte proliferation and combined use of interval exercise and rhG-CSF had a better effect.(10)Interval exercise had a similar effect as rhG-CSF on promoting the mRNA expression of NKX2-5 and GATA4,regenerating the infarcted heart,decreasing cardiomyocyte apoptosis,activatingFGF1-PI3K-AKT signal pathway and promoting cardiomyocyte proliferation in MI rats.Conclusions:1.Interval exercise with or without rhG-CSF treatment could promote BMCs mobilization to the circulation system and home to the infarct zone and peri-infarct region under the function of SDF-1 in MI rats.Combined use of interval exercise and G-CSF had a better effect;2.Interval exercise with or without rhG-CSF treatment could promote cardiomyocyte proliferation and decrease cardiomyocyte apoptosis,improve cardiomyocyte systolic and diastolic function and calcium transients,and promote cardiac function in MI rats;3.Exogenous injection of recombinant rat FGF1 could increase the FGF1 level in circulation system and myocardium,improve cardiomyocyte proliferation and inhibit cell apoptosis;4.FGF1-PI3K-AKT signal pathway was compensatory activated by MI;Interval exercise could activateFGF1-PI3K-AKT signal pathway and promote cardiomyocyte proliferation.Combined use of interval exercise and G-CSF had a better effect.These results indicated thatFGFl-PI3K-AKT signal pathway plays an important role in the improvement of cardiomyocyte proliferation and cardiac function by interval exercise and rhG-CSF treatment.