【作者】 申江峰；

【导师】 郑世营；

【作者基本信息】 苏州大学 ， 心胸外科学， 2010， 硕士

【摘要】 目的:研究促红细胞生成素(EPO)预处理在大鼠心肌缺血再灌注损伤中的保护作用与机制。方法:84只雄性SD大鼠随机分为3组:即正常组、缺血再灌注组(IR组)、EPO处理组(EP组)。EP组大鼠建立模型前24h腹腔注射促红细胞生成素5000U/kg做药物预处理,IR组建立模型前24h腹腔注射1ml生理盐水。心脏停搏后置于4℃St.ThomasII号停搏液中分别保存4h、6h、8h(各时间位点均为心脏6只),保存结束后制备活鼠替代Langendorff模型的灌注心脏模型。测定各组心脏保存前及保存后再灌注60min的血流动力学(HR、CF、LVDP、LVEDP、±dp/dtmax)等心功能指标,心肌酶(LDH、CK、CK-MB)释放量,心肌含水量。实验结束分别取各组左心室心肌标本,观察心肌常规和超微组织结构的变化。采用缺口末端标记(TUNEL)技术检测各组心肌细胞凋亡率。Western blot检测心肌组织中Caspase-3蛋白表达水平。结果:(1)心脏保存4h、6h、8h后再平衡灌注60min,EP组的心脏血流动力学及心功能各项指标恢复明显优于IR组(P<0.05或P<0.01)。(2)随着保存延长,各组血清CK、CK-MB及LDH水平逐渐升高。在各时间点CK、CK-MB和LDH水平,IR组高于EP组,差异有统计学意义(P<0.05或P<0.01)。(3)心脏保存8h,尽管实验组心功能各项指标有明显下降,但心脏仍全部复跳,IR组有3只心脏不能复跳。(4)TUNEL法检测结果显示:IR组及EP组凋亡指数均高于正常组,EP组心肌细胞凋亡数明显低于IR组。(5)Western blot检测结果显示:各组Caspase-3蛋白均有所表达,EP组Caspase-3蛋白表达量低于IR组(P<0.05),表达明显受到抑制。(6)光镜显示正常组心肌纤维排列规则,细胞核结构完整,为正常心肌组织结构。IR组心肌纤维排列紊乱,细胞核不规则,胞浆着色深,间质明显水肿,有炎症细胞浸润。EP组心肌组织损伤相对较轻,心肌纤维排列较规整,间质水肿不明显,间质内可见少量炎症细胞浸润。EP组及IR组光学显微镜下心肌组织均表现为不同程度的心肌空泡变性,伴有灶性心肌损害,炎性细胞浸润。随着保存时间的延长,衰变越明显。心脏保存8h后,IR组表现为弥漫性多类型细胞浸润,伴有明显的水肿、出血和心肌坏死,EP组较对照组明显减轻。(7)透射电镜显示空白组心肌细胞结构基本正常,肌原纤维平行排列,肌节清晰,线粒体形态一致,呈椭圆形,细胞核结构正常,而IR组和EP组均可见肌原纤维排列紊乱、断裂,线粒体肿胀或固缩,嵴溶解,部分线粒体空化。细胞核内染色质边集,细胞质水肿明显。EP组细胞结构损伤较轻,可见肌原纤维排列较规则,无明显溶解、断裂,线粒体形态接近正常,核内核仁清楚。结论:1、EPO预处理可以促进保存心脏心功能恢复,显著增强心肌收缩能力,改善顺应性。2、EPO预处理可以减轻心肌水肿、充血及渗出,血管扩张及炎症细胞浸润,减少细胞凋亡和Caspase-3基因表达,提高心脏保存质量,明显减轻心肌细胞缺血再灌注损伤。更多还原

【Abstract】 Objective:To study the protective effects and mechanisms of Erythropoietin preconditioning on myocardial ischemia-reperfusion injury (MIRI) in rats.Methods:84 male SD rats were randomly divided into 3 groups:Normal group、ischemia-reperfusion group (group IR)and erythropoietin preconditioning group(group EP)。The hearts of normal group were perfused without cardioplegia and heart storage.Rats of EP group were injected with EPO(5000U/Kg) in abdominal cavity 24 h prior to operation respectively,the rats of IR group were injected with nomal sodium.We made rat MIRI model through create a animal model using a living rat take place of Langendorff modle. The hearts were arrested with St.ThomasII and stored in the St.ThomasII solution.All hearts were stored for 4h,6h and 8h with 4℃hypothermia,and underwent 60 minutes of reperfusion.The parameters of hemodynamics (HR、CF、LVDP、LVEDP、±dp/dtmax),the myocardial enzymes(LDH、CK、CK-MB)were measured before and during reperfusion. Detecting the apoptosis index(AI) of myocardial cells at the end of experiment by TUNEL. The expression of Caspase-3 protein in cardiac myocytes were assayed by the methods of Western blot. Optical microscopy and transmission electron microscopy were used to observe the pathological changes of myocardial tissue and cells respectively.Results: (1)After the hearts were stored for 4h,6h,8h,the recovery of hemodynamics and cardiac function of the EP group were significantly better than the IR group during reperfusion (P<0.05 or P<0.01 ).(2)The myocardial enzymes leakage was appeared in every group,With the time past , the levels of the myocardial enzymes(LDH、CK、CK-MB) in serum is gradually increased ,the levels of the myocardial enzymes of the EP group was less than it the IR group.(P<0.05 or P<0.01()3)All the hearts of EP group could beat again during reperfusion, but the heart function decreased.But 3 hearts stored for 8 hours of IR group could not beat again.(4) The results of TUNEL showed that cardiomyocyte apoptosis index(AI) in the Control group and EP group were significantly less than IR group(P<0.05 or P<0.01()5)The results of Westem blot showed that the expression of Caspase-3 protein can be found in all time points.In the EP group,protein expression of Caspase-3 became obviously lower than IR group at all time point(P<0.05 or P<0.01). (6)By the optical microscopy, in normal group, myocardial fibre arranged in order,the structural integrity of the nucleus. The myocardial tissue of IR group showed extensive necrosis and severe muscle fiber fracture, myocardial cells dissolution and disappearance, neutrophils exudation. EP group displayed a reduced degree of myocardial neutrophilic infiltrate,necrosis, hemorrhage, and spindle-shaped interstitial cells .After ischemia and reperfusion myocardial samples were qualitatively assessed by transmission electron microscopy for structural changes. Under the TEM, IR group showed myofibrils disarrangement, large areas of myofibrils fracture, disappearance, sarcomere structure unclear, myocardial nuclear swelling, the nuclear membrane rupture, nuclear chromatin asymmetry, condensation, margination, mitochondrial morphological abnormalities and swelling, the rige disarrangement, fracture, disappearance, forming cavity, some mitochondria parceled by double membrane, and completely desquamated from the mother cells, forming apoptotic bodies in IR group. In EP groups, myocardial ultrastructural changes were discriminating. The structural damage in EP group was relatively less than IR group.We can see more unclear sarcomeres,no significant dissolution,no fracture,almost normal mitochondria and unclear nucleolus.Conclusion: (1)Erythropoietin preconditioning in rats can promote the preserved heart’s recovery of function, enhanced myocardial contractibility significantly,improved myocardial compliance.(2) Erythropoietin preconditioning can protect cardiomyocyte from ischemia reperfusion injure. The mechanism may be associated with that the Erythropoietin can anti-inflammatory, anti-oxidation, promote angiogenesis and reduce expression of caspase-3 gene and the occurrence of cardiomyocyte apoptosis.更多还原