Flow cytometric analysis of circulating microvesicles derived from myocardial ischemic preconditioning and cardioprotection of ischemia/reperfusion injury in rats  

作　　者：Miao LIU Yi-lu WANG Man SHANG Yao WANG Qi ZHANG Shao-xun WANG Su WEI Kun-wei ZHANG Chao LIU Yan-na WU Ming-lin LIU Jun-qiu SONG Yan-xia LIU 

机构地区：[1]Department of Pharmacology, School of Basic Medical Sciences, Tianjin Medical University [2]Section of Endocrinology, Department of Medicine, Temple University School of Medicine [3]Department of Dermatology, Perelman School of Medicine, University of Pennsylvania

出　　处：《中国应用生理学杂志》2015年第6期524-531,共8页Chinese Journal of Applied Physiology

基　　金：supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China(20101202110005);the Natural Science Foundation of Tianjin(11JCZDJC18300);the Research Foundation of Tianjin Municipal Education Commission(20110106)

摘　　要：Objective: To establish a flow cytometric method to detect the alteration of phenotypes and concentration of circulating microvesicles(MVs) from myocardial ischemic preconditioning(IPC) treated rats(IPC-MVs), and to investigate the effects of IPC-MVs on ischemia/reperfusion(I/R) injury in rats. Methods: Myocardial IPC was elicited by three cycles of 5-min ischemia and 5-min reperfusion of the left anterior descending(LAD) coronary artery. Platelet-free plasma(PFP) was isolated through two steps of centrifugation at room temperature from the peripheral blood, and IPC-MVs were isolated by ultracentrifugation from PFP. PFP was incubated with anti-CD61, anti-CD144, anti-CD45 and anti-Erythroid Cells, and added 1, 2 μm latex beads to calibrate and absolutely count by flow cytometry. For functional research, I/R injury was induced by 30-min ischemia and 120-min reperfusion of LAD. IPC-MVs 7 mg/kg were infused via the femoral vein in myocardial I/R injured rats. Mean arterial blood pressure(MAP), heart rate(HR) and ST-segment of electrocardiogram(ECG) were monitored throughout the experiment. Changes of myocardial morphology were observed after hematoxylin-eosin(HE) staining. The activity of plasma lactate dehydrogenase(LDH) was tested by Microplate Reader. Myocardial infarct size was measured by TTC staining. Results: Total IPC-MVs and different phenotypes, including platelet-derived MVs(PMVs), endothelial cell-derived MVs(EMVs), leucocyte-derived MVs(LMVs) and erythrocyte-derived MVs(RMVs) were all isolated which were identified membrane vesicles(<1 μm) with corresponding antibody positive. The numbers of PMVs, EMVs and RMVs were significantly increased in circulation of IPC treated rats(P<0.05, respectively). In addition, at the end of 120-min reperfusion in I/R injured rats, IPC-MVs markedly increased HR(P<0.01), decreased ST-segment and LDH activity(P<0.05, P<0.01). The damage of myocardium was obviously alleviated and myocardial infarct size was significantly lowered after IPC-MVs treatment(P<0.01). Conclusion: Objective： To establish a flow cytometric method to detect the alteration of phenotypes and concentration of circulating microvesicles（MVs） from myocardial ischemic preconditioning（IPC） treated rats（IPC-MVs）, and to investigate the effects of IPC-MVs on ischemia/reperfusion（I/R） injury in rats. Methods： Myocardial IPC was elicited by three cycles of 5-min ischemia and 5-min reperfusion of the left anterior descending（LAD） coronary artery. Platelet-free plasma（PFP） was isolated through two steps of centrifugation at room temperature from the peripheral blood, and IPC-MVs were isolated by ultracentrifugation from PFP. PFP was incubated with anti-CD61, anti-CD144, anti-CD45 and anti-Erythroid Cells, and added 1, 2 μm latex beads to calibrate and absolutely count by flow cytometry. For functional research, I/R injury was induced by 30-min ischemia and 120-min reperfusion of LAD. IPC-MVs 7 mg/kg were infused via the femoral vein in myocardial I/R injured rats. Mean arterial blood pressure（MAP）, heart rate（HR） and ST-segment of electrocardiogram（ECG） were monitored throughout the experiment. Changes of myocardial morphology were observed after hematoxylin-eosin（HE） staining. The activity of plasma lactate dehydrogenase（LDH） was tested by Microplate Reader. Myocardial infarct size was measured by TTC staining. Results： Total IPC-MVs and different phenotypes, including platelet-derived MVs（PMVs）, endothelial cell-derived MVs（EMVs）, leucocyte-derived MVs（LMVs） and erythrocyte-derived MVs（RMVs） were all isolated which were identified membrane vesicles（〈1 μm） with corresponding antibody positive. The numbers of PMVs, EMVs and RMVs were significantly increased in circulation of IPC treated rats（P〈0.05, respectively）. In addition, at the end of 120-min reperfusion in I/R injured rats, IPC-MVs markedly increased HR（P〈0.01）, decreased ST-segment and LDH activity（P〈0.05, P〈0.01）. The damage of myocardium was obviously alleviated and myo

关 键 词：缺血/再灌注损伤 流式细胞仪分析 心肌梗死 缺血预处理 保护作用 大鼠 循环 微泡 

分 类 号：Q463[生物学—生理学] S476.13[农业科学—农业昆虫与害虫防治]