【作者】 王孝东；

【导师】 谷天祥；

【作者基本信息】 中国医科大学 ， 外科学， 2010， 博士

【摘要】 前言心肌梗死是威胁人类健康的重要心血管疾病之一,是引起猝死和心力衰竭的主要原因,所以有必要进行积极有效的再灌注治疗,以挽救濒死心肌。再灌注的形式,包括心血管介入治疗,冠状动脉搭桥,干细胞移植等,其中心脏的侧枝循环形成对于防止心肌梗死,心功能的保护具有重要意义。Apelin是1998年日本学者Tatemoto从牛胃组织提取物中分离出一种小分子多肽,是血管紧张素受体AT1相关受体蛋白(Angiotension II Protein J, APJ)的内源性配体,Apelin/APJ信号通路可以促进细胞内多种信号通路的激活产生多种生理功能。Apelin在心血管系统高度表达,它具有增强心肌收缩力、扩张血管、减轻心脏负荷、促进血管生长、调节水盐平衡等作用,其中Apelin促进血管形成的作用,越来越引起学者的重视。Apelin信号对心脏和血管的形成有重要作用。它在视网膜血管的内皮层中也高度表达,Apelin是一个视网膜内皮细胞的血管生成因子。与许多其它的血管生成因子相似,在低氧的情况下,Apelin基因的表达是增加的。用反义技术抑制Apelin的表达会导致血管生成障碍,Apelin具有促有丝分裂、促进分化和抑制凋亡的作用。另外,Apelin及其受体Agtrlb还控制斑马鱼原肠时期心脏形态的发生Apelin和Agtrllb受体表达不足或者表达过度都将导致心脏前体缺陷并最终累及心脏发育。另外,Apelin会加速血管生成开关的打开,潜在的激活了肿瘤的新生血管形成。Apelin基因在三分之一的人类肿瘤中表达上调。Apelin促进新生血管的特性,已经在小鼠的肿瘤新生血管形成过程中得到证实。Apelin对内皮细胞的促有丝分裂和血管生成的特性意味着它也许在治疗与血管生成有关的疾病方面有潜在的作用。APJ的受体激动剂或许能够作为药理学工具在血管形成对抗缺血性疾病方面有治疗意义,而它的拮抗剂或许能够阻止血管形成,以阻止肿瘤生长和缺血性视网膜病。Apelin作为一个强有力的内皮生长和血管生成的刺激因子,激活这种信号通路,能否促进缺血心肌的血管生成,减轻心肌缺血所致的损伤或者坏死,目前尚不得而知。本课题旨在通过观察：Apelin是否参与和保护心肌缺血损伤及其机制；外源性给予Apelin能否促进内皮细胞的增殖,迁移和血管形成；外源性给予Apelin毙否增加心肌细胞侧支循环的形成,Apelin是通过那个通路促进血管的形成的,从而为挽救缺血、濒死的心肌寻找一条有效的途径。目的1、建立在体大鼠心肌缺血模型,观察心肌缺血时Apelin是否对心脏有保护作用及其机制。2、外源性给予Apelin能否促进心脏侧支循环的形成,减少心肌缺血或者心肌梗死。3、通过HUVECs了解Apelin对内皮细胞增殖,迁移和血管形成的影响,以探讨Apelin促进血管形成的可能机制,以及这种机制是否与哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)转导通路相关。方法1、Apelin对心肌缺血大鼠的心脏保护作用(1)雄性Sprague-Dawley (SD)大鼠左冠状动脉前降支结扎,建立心肌缺血模型。腹腔注射Apelin (1μg/kg.d×2),应用心导管检查心率(HR)、左室最大收缩压(LVESP)、左室舒张末期压(LVEDP)、左室内压最大升降速度(±P/dTmax)以评价Apelin对心功能的影响。(2)雄性SD大鼠冠状动脉前降支结扎,建立心肌缺血模型,腹腔注射Apelin (1μg/kg.d×2),抽血检测血中乳酸脱氢酶(Lactate dehydrogenase, LDH)含量。2、Apelin对心肌缺血大鼠心脏侧枝循环的影响(1)大鼠冠状动脉前降支结扎,建立心肌缺血模型,同时给予Apelin腹腔注射(1μg/kg.d×7)以观察Apelin对心脏侧枝循环的影响。(2)酶联免疫法(enzyme linked immunosorbent assay, ELISA)测定大鼠心肌缺血损伤时血小板-内皮细胞粘附分子1(PECAM1/CD31)蛋白含量。(3)伊文氏蓝和红四氮唑(TTC)双染色,应用计算机图象分析系统计算心肌梗死面积。(4)通过CD31免疫组化染色,在显微镜下观察缺血心肌组织的血管生成情况。(5)HE染色显微镜下观察缺血心肌组织的损伤情况。(6)通过western blot检测VEGF和CD31的表达。3、Apelin对内皮细胞增殖,迁移和血管形成的影响(1)应用甲基噻唑基四唑(methyl thiazolyl tetrazolium, MTT)的方法观察Apelin对HUVECs增殖的影响。(2)通过Transwell观察Apelin对HUVECs迁移的影响。(3)应用Tubeformation观察Apelin对HUVECs血管形成的影响。结果1、Apelin对心肌缺血大鼠心功能的保护作用与其它各组比较,大鼠心肌缺血48h时Apelin治疗组(1μg／kg.d×2,腹腔注射)能明显提高LV±dp/dtmax、LVESP,降低LVEDP(P<0.05、0.01)。2、Apelin对心肌缺血大鼠心肌酶的影响自动生化仪结果显示,大鼠心肌缺血48h时Apelin治疗组(1μg/kg.d×2,腹腔注射)的LDH含量明显较其它各组为低(P<0.01)。3、Apelin对心肌缺血大鼠CD31表达的影响酶联免疫法(ELISA)测定大鼠心肌缺血大鼠的CD31结果显示,Apelin治疗组(1μg／kg.d×7,腹腔注射)的CD31含量明显较其它各组为高(P<0.05)。4、伊文氏蓝和红四氮唑(TTC)双染色,通过计算机图象分析系统统计心肌梗死面积结果发现,Apelin治疗组(1μg／kg.d×7,腹腔注射)的心肌梗塞面积明显较其它各组为低(P<0.05)。5、Apelin对心肌缺血大鼠微血管密度的影响CD31免疫组化染色,显微镜下观察心肌组织的微血管密度(MVD, microvessel density)发现,Apelin治疗组的MVD计数明显较其它各组为多(P<0.01)。6、Apelin对心肌缺血大鼠心肌结构的影响HE染色显微镜下观察发现Apelin治疗组的瘢痕形成较其它各组少。7、Apelin对心肌缺血大鼠VEGF和CD31表达的影响Western blot结果显示Apelin组(1μg／kg.d×7,腹腔注射)的VEGF和CD31蛋白表达明显较其它各组为高。8、Apelin对HUVECs增殖的影响应用MTT观察Apelin对HUVECs增殖的影响发现,与其它各组相比Apelin (Apelin lumol/L)可以明显的促进HUVECs的增值(P<0.01)9、Apelin对HUVECs迁移的影响应用Transwell观察Apelin对HUVECs迁移的影响发现,与其它各组相比Apelin (Apelin 1μg/ml)可以明显的促进HUVECs的迁移(P<0.01)。10、Apelin对HUVECs血管形成的影响Tubeformation观察Apelin对内皮细胞血管形成的影响发现,Apelin (Apelin 60uM)治疗组的小管形成的数目较其他各组为多(P<0.05)。11、尽管Apelin对缺血心肌和HUVECs有上述作用,但是在应用了PLC的抑制剂U-73122以后,Apelin的心肌保护和心功能保护作用消失；在应用了mTOR的阻断剂-雷帕霉素后,Apelin对心肌缺血侧枝循环和对HUVECs促进增殖、迁移和血管形成的作用消失,与对照组无明显差异(P>0.05)。结论1、Apelin可以保护心肌缺血的心脏损伤和心功能。2、Apelin保护心肌缺血的心脏损伤和增强心肌收缩力可能与激活细胞内的PLC信号通路有关。3、Apelin增加心肌表达CD31,并可刺激心肌缺血区的血管生成,减少心肌梗死面积,促进心脏侧枝循环的形成。4、Apelin可以促进内皮细胞的迁移、增殖和血管形成。5、Apelin的促进缺血心肌侧枝循环和HUVECs血管的形成可能是通过激动mTOR受体发挥作用的,这些作用可以被mTOR的拮抗剂-雷帕霉素所阻断。更多还原

【Abstract】 IntroductionAcute myocardial infarction is one of most important disease threatening human life and health. It is the chief reasons of sudden death and heart failure. It is necessary to reperfute myocardium in order to save dying myocardium. There are many kinds of reperfusions, including nterventional therapy, coronary artery bybass graft, stem cell transplantation and so on. Among of these ways the collateral circulation formation is very important for preventing myocardial infarction and protecting heart functons. Apelin is a micromolecule polypeptide which has several effects on cardiovascular system. It was separated from bovine stomach by Tatemoto in 1998 and it is the endogenic ligand of angiotensionⅡprotein J (APJ). The Apelin/APJ could activate many intracellar signals and produce several physical functions. Apelin is expressed in cardiovascular system extensively. The Apelin/APJ system can reinforce myocardial contractile force, relax blood vessels, relieve cardiac loads, promote blood vessels growth and regulate body fluid equilibrium etc. Among theses effects the character of Apelin to promote blood vessels growth is more and more attracting interests to hakeems.Apelin signal has important effects on formation of blood vessels and hearts. And Apelin was found to express in retinal vessels endothelium extensively and Apelin can be a angiogenesis factor for retina endotheliocytes. Apelin expression is aμgmented under mionectic circumstances just like other angiogenesis factors. The vascularization would be dyspoiesis if Apelin expression was blocked with antisense technique. Apelin had effects of promoting karyokinesis, differentiation and inhibiting apoptosis. Apelin and its receptor, agtrllb, controlled the genesis of zebra-fish heart. Apelin and its receptor would effect heart development whether their expression was deficient or excessive. Moreover, Apelin would open the angiogenic switch and activate tumor neovascularization. Apelin is increased in one-third of human tumors. The character of Apelin enhancing neovascularization has been confirmed in mice neovascularization.Apelin has the character.of promoting endotheliocytes karyokinesis and angiogenesis, which means it could have the potent character of curing diseases of related angiogenesis. The APJ receptor agonist would treat ischemic diseases through angiogenesis while the APJ receptor blocker would prevent tumors growth and retinopathy. However, it is not reported that as a powerful factor of promoting endotheliocytes proliferation and angiogenesis whether or not Apelin could promote the angiogenesis of ischemic myocardium and lessen the injury or necrosis induced by ischemia.The aims of the study were to explore:if Apelin could take part in and protect myocardial ischemia injury; if Apelin given exogenously could promote proliferation, migration and angiogenesis of endotheliocytes; if Apelin given exogenously could enchance angiogenesis of ischemic hearts and on which pathway Apelin promote angiogenesis in order to find an effective way to save ischemic and dying myocardium.Objective1. To observe if Apelin could protect ischimic heart by setting up a rat myocardium ischemic model;2. To confirm if Apelin given exogenously could promote heart collateral circulation and reduce the injury of myocardil ischemia and myocardial infarction;3. To study the effects of Apelin on the proliferation, migration and tubeformation of HUVECs; To explore the possible mechanisms of Apelin promoting angiopoiesis and if these mechanisms have relations to Apelin and mTOR.Methods1. To detect the protcetion effects of Apelin on ischimic rat.(1) To ligate the male SD rats left anterior descending (LAD) coronary artery and set up a myocardial ischimia model. Apelin was given(intraperitoneal injection, 1μg/kg.d×2) to examine heart rate (HR), left ventricular end-diastolic pressure(LVEDP), left ventricular end-systolic pressure (LVESP) and maximal left ventricle developed pressure (LV±dp/dtmax) with a cardiac catheterization in order to evalue the effects of Apelin on heart function. (2) Apelin was given (intraperitoneal injection, lμg/kg.d×2) to detect the content of LDH by ligating the male SD rats’LAD and a ischemic model.2. To examine the effects of Apelin on the collateral circulation of ischemic rat hearts.(1) To ligate the male SD rats’left anterior descending (LAD) coronary artery and set up a myocardial ischimia model. Apelin was given (intraperitoneal injection, 1μg/kg.d×7) to observe the.effects of Apelin on collateral circulation in ischemic rats.(2) To detect the content of CD31 in the myocardium of ischemic rats with enzyme linked immunosorbent assay (ELISA).(3) To dye with Azo-Blue and TTC and to calculate the myocardial infarction aera with a image analysis system (IAS).(4) To observe the angiogenesis ischemic of myocardium dyed with CD31 immunohistochemistry under a microscope.(5) To observe the injury of ischemic myocardium dyed with HE under a microscope.(6) To oberve the protein expression of VEGF and CD31with western blot.3. To examine the effects of Apelin on the proliferation, migration and angiopoiesis of HUVECs.(1) To observe the effect of Apelin on the proliferation of HUVECs by methyl thiazolyl tetrazolium (MTT).(2) To explore the effect of Apelin on the migration of HUVECs with Transwell.(3) To study the effect of Apelin on the angiopoiesis of HUVECs with tubeformation.Results1. The effects of Apelin on heart function of myocardial ischemia in rats The group of Apelin (1μg/kg.d×2, intraperitoneal injection) enchanced LV±dp/dtmax、LVESP and reduced LVEDP significantly compared with other groups at ischemia 48h (P<0.05、0.01).2. The effects of Apelin on myocardial enzymes of myocardial ischemia in rats The Apelin group (1μg/kg.d×2, intraperitoneal injection) was lower LDH significantly compared with other groups at ischemia 48h showed by automatic biochemistry machine (P<0.01).3. Apelin had effects on CD31 content of myocardial ischemia in rats ELISA of rat CD31 showed that Apelin group (1μg/kg.d×7, intraperitoneal injection) was higher compared with other groups (P<0.05).4. The effects of Apelin on myocardial anfarction of myocardial ischemia in rats The results of infarction aera dyed with Azo-Blue and TTC and calculated with IAS discovered that Apelin group (1μg/kg.d×7, intraperitoneal injection) was significantly lower compared with other groups (P<0.05).5. The effects of Apelin on MVD of myocardial ischemia in rats The CD31immunohistochemistry dye showed that the MVD of Apelin group (1μg/kg.d×7, intraperitoneal injection) was significantly higher compared with other groups under microscope (P<0.01).6. The effects of Apelin on myocardial construction of myocardial ischemia in rats The HE dye showed that the scar of Apelin group (1μg/kg.d×7, intraperitoneal injection) were significantly much less compared with other groups under microscope.7. The effects of Apelin on VEGF and CD31 expression of myocardial ischemia in rats The expression of VEGF and CD31 were higher significantly compared with other groups (P<0.01).8. The effects of Apelin on the proliferation of HUVECs The proliferation of Apelin on HUVECs with MTT showed that Apelin group (Apelin lumol/L) could promote proliferation of HUVECs significantly compared with other groups (P<0.01).9. The effects of Apelin on the migration of HUVECs The migration of Apelin on HUVECs with transwell showed that.Apelin group(Apelin 1μg/mL) could promote migration of HUVECs compared with other groups (P<0.01).10. The effects of Apelin on the angiopoiesis of HUVECs The angiopoiesis of Apelin on HUVECs with tubeformation showed that.Apelin group(Apelin 60uM) could promote angiopoiesis of HUVECs compared with other groups (P<0.05).11. Although Apelin had effects above mentioned the effects, however, the effects of Apelin on myocardial protection and cardiac function in ischemic rats disappered when Apelin was adminstered with U-73122 simultaneously. And the effects of Apelin on collateral circulation in ischemic rats and proliferation, migration and tubeformation in HUVECs were disappeared when Apelin was adminstered with rapamycin simultaneously.Conclusions1. Apelin could protect cardiac function and injury in ischemic rat hearts.2. Apelin protected cardiac function and injury in ischemic rat hearts by acting PLC signal pathway.3. Apelin could promote myocardium to expression CD31, stimulate angiogenesis, reduce myocardial infarction area and enchance collateral circulation in ischemic myocardium.4. Apelin could facilitate prolifeation, migration and tubeformation in HUVECs.5. Apelin could promote collateral circulation in ischemic hearts and tubeformation in HUVECs by exciting mTOR receptors and could be blocked by the antagon of mTOR-rapamycin.更多还原