间歇运动和粒细胞集落刺激因子促进心梗大鼠干细胞动员与内源性心肌细胞增殖的激光共聚焦/流式细胞术观察分析

The Observation and Analysis of Stem Cell Mobilization and Endogenous Cardiomyocyte Proliferation Promoted by Interval Exercise and Granulocyte Colony-stimulating Factor Using Confocal Microscopy and Flow Cytometry in Myocardial Infarction Rats

目的:探讨跑台间歇运动结合粒细胞集落刺激因子对心肌梗死大鼠干细胞动员的效果和在内源性心肌细胞增殖中的作用及其对心功能的影响。方法:90只成体雄性SD大鼠随机分为假手术组(Sham)、心梗组(MI)、心梗+间歇运动组(ME)、心梗+粒细胞集落刺激因子(G-CSF)组(MG)和心梗+G-CSF+间歇运动组(MGE)。结扎大鼠左冠状动脉前降支制备心肌梗死模型。ME和MGE组大鼠在心肌梗死手术结束1周后进行3周跑台间歇运动。MG和MGE组大鼠术后1h皮下注射人源重组粒细胞集落刺激因子(rhG-CSF),10μg/kg/d×5d,其他各组大鼠给予同剂量生理盐水。免疫荧光法检测并计算梗死边缘区心肌细胞增殖百分率;流式细胞术检测外周血单个核细胞中c-kit^+和CD29^+细胞百分率;Western Blot方法检测心肌组织中c-kit和CD29蛋白表达水平。TTC染色检测心肌梗死面积百分率;彩色多普勒超声和血流动力学方法检测心功能。结果:与Sham组相比,MI组PCNA^+心肌细胞百分率、外周血单个核细胞中c-kit^+和CD29^+细胞百分率、心肌组织中c-kit和CD29蛋白表达和心肌梗死面积显著增加,心功能显著降低;与MI组比较,ME、MG和MGE组大鼠梗死边缘区心肌组织中PCNA^+心肌细胞百分率、外周血单个核细胞中c-kit^+和CD29^+细胞百分率、心肌组织中c-kit和CD29蛋白表达均显著增加,梗死面积显著减小,心功能显著增强,且MGE组变化最为显著。结论:间歇运动或粒细胞集落刺激因子可显著促进干细胞动员归巢,诱导心肌细胞增殖,缩小梗死面积,提升心功能,且二者联合效果优于单一因素作用。

Objectives：To discuss the effects of treadmill interval exercise and granulocyte colony-stimulating factor（G-CSF）on stem cell mobilization,endogenous cardiomyocyte proliferation and cardiac function in rats with myocardial infarction（MI）.Methods：90 Adult male sprague-dawley rats were randomly divided into：Sham-operated group（Sham）,MI group（MI）,MI with interval exercise group（ME）,MI with G-CSF treatment group（MG）and MI with G-CSF treatment plus interval exercise group（MGE）.The MI model of rats was established by ligation of the left anterior descending（LAD）coronary artery.Rats in ME and MGE groups were subjected to 3-week treadmill interval exercise seven days after myocardial infarction.Rats in MG and MGE groups were injected with rhG-CSF subcutaneously 1h after myocardial infarction,10μg/kg/d for 5 days.Cardiomyocyte proliferation ratio was detected by immunofluorescence and calculated in the peri-infarct region.C-kit and CD29 positive peripheral blood mononuclear cells（PBMCs）were counted by flow cytometer.C-kit and CD29 expression in peri-infarct area was measured by western blot.Hearts were picked for TTC dyeing to decide the myocardial infarction area of each group.Hemodynamic measurement and echocardiography were performed to evaluate cardiac function.Results：Compared to Sham group,the ratio of PCNA and cTnT dual positive cardiomyocytes,c-kit~＋and CD29~＋ cells ratio in the PBMCs,c-kit and CD29 expression in the peri-infarct region,myocardial infarction area were significantly increased and cardiac performance decreased significantly in MI rats;compared to MI rats in periinfarct region,percentage of PCNA and cTnT dual positive cardiomyocytes,c-kit and CD29 positive PBMCs ratio,the expression of c-kit and CD29 protein in the peri-infarct region,cardiac performance were increased significantly,and myocardial infarction area decreased significantly in ME,MG and MGE group.The combined treatment with G-CSF and interval exercise had a better effect than either of them.Conclusions：Interval exercise or G-CSF could promote stem cell mobilization and homing,cardiomyocyte proliferation,cardiac performance and decrease myocardial infarction area significantly in MI rats.The better effects were shown with the combined treatment.