有氧运动对心肌细胞增殖的影响及CBP的调控作用

Effect of Aerobic Exercise on Cardiomyocyte Proliferation and Regulation of CBP

目的:探讨有氧运动对心肌细胞增殖的激活和cAMP反应元件结合蛋白绑定蛋白(CBP)在其中的调控作用。方法:采用游泳训练(Swim)2周构建运动性心肌肥大模型,采用主动脉弓缩窄术(TAC)2周构建病理性心肌肥大模型。12周龄雄性C57Bl/6J小鼠随机分为4组:对照组(Control)、运动性心肌肥大组(Swim)、假手术组(Sham)、病理性心肌肥大组(TAC),每组10只。实验结束后,采用二维超声心动图检测室间隔厚度(IVS)、左室后壁厚度(LVPW)和心功能。对心肌组织蛋白采用Western Blot检测增殖细胞核抗原(PCNA)的表达水平,衡量心肌细胞增殖能力。采用RT-PCR、Western Blot和免疫组化的方法检测心肌细胞中CBP的表达水平。为验证CBP对心肌细胞增殖能力的激活作用,在离体实验中,以去氧肾上腺素(PE)诱导原代心肌细胞产生肥大,采用CBP抑制剂C646抑制CBP表达。离体实验分组情况:1)对照组(Control);2)PE组;3)C646组;4)PE+C646组。对心肌细胞爬片进行α-肌动蛋白(α-actin)的免疫荧光实验检测心肌细胞大小,免疫荧光检测增殖细胞核抗原Ki67的表达衡量心肌细胞增殖水平,RT-PCR检测CBP的mRNA水平。结果:1)游泳运动和TAC均能使小鼠产生明显的心肌肥大,与相应的对照组相比,两种心肌肥大模型的IVS、LVPW、左室容量(LV Vol)和射血分数(EF)都有明显升高(P<0.05)。2)Swim组小鼠心肌组织PCNA蛋白表达水平明显高于Control组(P<0.05)和TAC组(P<0.01)。3)Swim组心肌组织CBP的mRNA水平比Control组明显升高(P<0.01),且明显高于TAC组(P<0.05)。Swim组心肌组织CBP蛋白表达水平与Control组相比有明显升高(P<0.05),且显著高于TAC组(P<0.01),TAC组比Sham组明显降低(P<0.05)。心肌组织切片CBP的免疫组化结果显示,Swim组CBP阳性细胞显著多于Control组(P<0.01)与TAC组(P<0.01)。4)离体实验结果显示,PE组心肌细胞面积显著高于Control组(P<0.05)和PE+C646组(P<0.05)。PE能使CBP的mRNA表达水平比Control组明显升高(P<0.01),PE+C646组CBP的mRNA水平比PE组明显降低(P<0.01)。PE组Ki67阳性细胞与Control组相比明显增多(P<0.01),PE+C646组Ki67阳性细胞与PE组相比明显减少(P<0.05)。结论:有氧运动能明显激活心肌细胞的增殖能力,并伴随着CBP的mRNA和蛋白表达水平升高,抑制CBP的表达水平可以抑制心肌细胞的增殖能力,提示,CBP在有氧运动促进心肌细胞增殖的过程中发挥着重要的调控作用。

Objective： To explore the effect of aerobic exercise on the activation of cardiomyocytc proliferation in cardiac hypertrophy and the role of cyclic-AMP-responsive element binding protein binding protein （CBP） in it by in vlvo and in vitro experiments. Method： The cxcrcscinduced cardiac hypertrophy model was made by swim training for 2 wks, and the pathological model was made by transverse aortic constriction （TAC） for 2 wks. 12-week-old C57Bl/6J mice were divided randomly into control group, swim group, sham group and transverse aortic constriction （TAC） group. By the end of experiments, cardiac hypertrophy, contractility were evaluated by echocardiography. The expression level of myocardial proliferating cell nuclear antigen （PCNA） were examined by Western Blot to evaluate the degree of cardiomyocytes proliferation. The expression level of myocardial CBP were examined by RT-PCR, Western Blot and immunohistochemical methods. To verify the effect of CBP on activation of cardiomyocytes pro liferation, we did in vitro experiment on primary cardiomyocytes. In this experimcnt, cardio myocyte hypertrophy was induced by phenylephrine （PE）, and C646 was used to inhibit the expression of CBP. The primary cardiomyocytes were divided randomly into 4 groulxs in vitro, control, PE, C646 and PE-b C646 groups. Meanwhile, eardiomyocyte hypertrophy was detected by a-actin immunofluoreseence, CBP mRNA expression was detected by RT-PCR, and the ex- pression level of Ki67 was detected by immunofluoreseence to evaluate tile degree of cardio- myocytes proliferation. Results, 1） Compared with corresponding control groups, inter ventric ular septum thickness （IVS）, left ventricular posterior wall thickness （LVPW）, left ventricle volume （LV Vol） and ejection factor （EF） of exercise-induced and pathological cardiac hypertrophy models were significantly increased （P〈0. 05）. 2） The protein expreion level of PC- NA in swim group was higher than that of control group （P〈0. 05） and TAC group （P〈2 0. 01）. 3） CBP mRNA expression of swim group was increased significantly than that of con trol （P〈0.01） and TAC group （P〈0. 05）. CBP protein expression of TAC was decreased significantly than that of Sham （P〈0.05）, and the level of swim were higher obviously than those of control （P〈0. 05） and TAC （P〈0. 01）. The immunohistochemlstry of CBP showed that CBP-positive cells of Swim was significantly more than Control （P〈0. 01） and TAC group （P〈0. 01）. 4） The results of in vitro experiment were that primary cardiomyocytes performance hypertrophy induced by PE （P〈0. 05）, and use of C646 could decrea cardiomyocyte hypertrophy （P〈0. 05） significangtly than that of PE. CBP mRNA expression of PE was significantly increased compared with Control （P〈0.05）, and this level was obviously inhibited by C646 （P〈0. 01）. The immunofluorescence of Ki67 showed that PE could increase Ki67-positive cells obviously （P〈0. 01）, and cardiomyocytes proliferation were inhibited （P 〈0.05） when CBP activity were suppressed. Conclusion： Aerobic exercise could promote myo- cardial proliferation through activating CBP. Cardiomyocytes proliferation was inhibited when CBP activity were suppressed. CBP plays an important role in the process of aerobic exercise promote cardiomyocytes proliferation.