1、目 录一、搁 璺中文论著摘要1英文论著摘要5：_二、英文缩略语10三、论文卤，苦j12日U舌j一1论文一Apelin对心肌梗死大鼠的心肌保护作用白，言16刖茜1材料与方法17结果18讨论19结论2l论文二Apelin对心肌缺血大鼠心脏侧枝循环的影响Ht言22 I画材料与方法22结果26讨论30结论33论文三Apelin对内皮细胞增殖，迁移和血管形成的影响币f。言34刚舌。1材料与方法34结果36讨论40结论42四、本研究创新性的自我评价43五、参考文献44六、附录综述48在学期间科研成绩71致谢72个人简介：”中文论著摘要-Apelin对心肌缺血大鼠心脏侧枝循环的影响刖 盂心肌梗死是威胁人类健
2、康的重要心血管疾病之一，是引起猝死和心力衰竭的ji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il：的肜式，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冉一种小分予多肽，是m管紧张素受体ATl相关受体蛋Ft(Angiotension II Protein J，APJ)的内源性配体，ApelinAPJ信号通路可以促进细胞内多种信号通路的激活产生多利，!l：-Eq!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
3、心肌!负1：；：、促进血管，卜长、调节水盐平衡等作用，其中Apelin促进m管形成的fi-Jfj，越水越引起学者的重视。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足一个视网膜内皮细胞的血管生成因子。与许多其它的血箭，i成1人l子相似，在低氧的情况一F，Apelin基凶的表达是增加的。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A甜rlb还控制斑马鱼原肠时期心脏形态的发乍，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祟及心肼
4、!发育。乡j外，Apelin会加速血管生成开关的打开，潜在的激活了肿瘤的新_!m篱彤成。Apelin基冈在三分之一的人类肿瘤中表达上调。Apelin促进新7L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jfIL管乍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Ij具在皿管形成对抗缺血性疾病方面有治疗意义，而它的拈抗剂或许能够阻J卜mL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7J的内皮生K衣J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，E成，减轻心肌缺血所
5、致的损伤或者坏死，目自仃尚不得而知。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3Apellm把甭促进内皮细胞的增贿，迁移和血管形成；外源性给予Apehnn一目i“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，从向为挽牧缺血、濒死的心肌寻找一条有效的途径。目 的1、建立在体火鼠心肌缺血模型，观察心肌缺血时Apelin足否对心脏自保护作用及其机制。2、外源性给予Apehn艇,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HUVECsT解Apelin对内皮细胞增殖，迁移和血管形成的影响，以探bApelin促进m管形成的可能机制
6、，以及这种机制是否与哺乳动物雷帕霉索靶蛋t：t(mammalian target of rapamycinmTOR)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SpragueDawley(SD)大鼠左冠状动脉前降支结扎，建锣心肌缺血模型。腹腔注射Apelin(199kgd2)，应用心导管检查心率(HR)、A窀最大收缩J丘(LVESP)、左室舒张木期妪(LVEDP)、左室内压最大升降速度(士dPdTm烈)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(199kgd2)，抽血检测血中乳酸脱氢酶(Lactat
7、e dehydrogenase，LDH)含：最。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(1“g瓜gdx7)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m损伤时血小板内皮细胞粘附分子l(PECAMlCD31)蛋自含量。(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2肌梗夕E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牛成情况。(5)HE染色显微镜下观察缺血
8、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)应用甲基噻哗基四哗(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(1l上gkgdx2，腹腔注射)能明显提高LV+dpdtm舯LV
9、ESP，降低LVEDP(尸1305、001)。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(11，Lgkgdx2，腹腔注射)的LDH含量明显较其它各组为低(Po01)。3、Apelin对心肌缺血大鼠CD31表达的影响酶联免疫法(ELISA)测定人鼠心肌缺血大鼠的CD31结果显示，Apelin治疗组(11tgkgdx7，腹腔注射)的CD31含量明显较其它各组为高(P锄05)。4、伊文氏蓝和红四氮哗(TTC)双染色，通过计算机图象分析系统统计心肌梗夕E面秘结果发现，Apelin治疗组(199kgdx7，腹腔注射)的心肌梗塞衙积明湿较其它各组为
10、低(尸005)。5、Apelin对心肌缺血大鼠微血管密度的影响CD31免疫组化染色，艋微镜下观察心肌组织的微血管密度(MVD，microvessel density)发现，Apelin治，组的MVD计数明显较其它各组为多(尸锄01)。6、Apelin对心肌缺血大鼠心肌结构的影响HE染色显微镜F观察发现Apelin治，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结果鼹乃Apelin组(199kgdx7，腹腔注射)的VEGF和CD31蛋白表达明显较其它各纽为由fuJ O8、Apelin对HUVECs增殖的影响应用MTT观察Apelin
11、对HUVECs增殖的影响发现，与其它各组相比Apelin(Apelin lumolL)可以明显的促进HUVECs的增值(P锄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 lggm1)可以明显的促进HUVECs的迁移(P001)。10、Apelin对HUVECs血管形成的影响Tubeformation观察Apelin对内皮细胞m管形成的影响发现，Apelin(Apelin 60uM)治疗组的小管形成的数目较，e他各纠1为多(JPo05)。11、尽管Apelin对缺血心肌和HUVECs有
12、上述作用，但足在应用了PLC的抑制荆U73122以后，Apelin的心肌保护和心功能保护作用消失；在应用了roTOR的阻断剂雷帕霉素后，Apelin对心肌缺血侧枝循环和对HUVECs促进增贿、迁移和JflL管形成的作用消失，与对照组无明显差异(胗005)。结论l、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PI。C信号通路有关。3、Apelin增加心肌表达CD31，并町刺激心肌缺血区的血管生成，减少心肌搜夕匕If；I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
13、进缺血心肌侧枝循环和HUVECs血管的形成可能足通过激动roTOR受体发挥作用的，这些作用可以被mTOR的拈抗剂雷帕霉素所阻断。关键词Apelin；心肌缺血；HUVECs；VEGF蛋白；CD31蛋白：侧枝循环；信号传导4英文论著摘要The Effects ofApelin Oil Myocardial Ischemia andCardiac Collateral CirculationIntroductionAcme myocardial infarction is one of most important disease threatening humanlife and healthIt
14、 is the chief reasons of sudden death and heart failureIt is necessaryto reperfute myocardium in order to save dying myocardiumThere are many kinds ofreperfusions，including nterventional ther印y，coronary artery bybass graft，stem celltransplantation and SO onAmong of these ways the collateral circulat
15、ion formation isvery important for preventing myocardial infarction and protecting heart functonsApelin is a micromolecule polypeptide which has several effects on cardiovascularsvstemIt WaS separated from bovine stomach by Tatemoto in 1 998 and it is theendogenic ligand of angiotension II protein J
16、(APJ)The ApelinAPJ could activatemany intracellar signals and produce several physical functionsApelin is expressedin cardiovascular system extensivelyThe ApelinAPJ system Can reinforce myocardialcontr乏Lctile forcerelax blood vessels，relieve cardiac loads，promote blood vesselsgrowth and regulate bod
17、y fluid equilibrium etcAmong theses effects the character ofApelin to promote blood vessels growth is more and more attracting interests tohakeemsApelin signal has important effects on formation of blood vessels and heartsAndApelin Was found to express in retinal vessels endothelium extensively and
18、Apelin canbe a angiogenesis factor for retina endotheliocytesApelin expression is aItgmentedunder mionectic circumstances just like other angiogenesis factorsThevascularization would be dyspoiesis if Apelin expression Was blocked with antisensetechniqueApelin had effects of promoting karyokinesis，di
19、fferentiation and inhibitingapoptosisApelin and its receptor，agtrllb，controlled the genesis of zebra-fish heartApelin and its receptor would effect heart development whether their expression wasdeficient or excessiveMoreover，Apelin would open the angiogenic switch andactivate tumor neovascularizatio
20、nApelin iS increased in one-third of human tumorsThe character of Apelin enhancing neovascularization has been confirmed in miceneovascularizationApelin has the character of promoting endotheliocytes karyokinesis andangiogenesis，which means it could have the potent character of curingdiseases ofrela
21、ted angiogenesisThe APJ receptor agonist would treat ischemic diseases throughangiogenesis while the APJ receptor blocker would prevent tumors growthandretinopathyHowever，it is not reported that as a powerful factor of promotmgendotheliocytes proliferation and angiogenesis whether or not Apelin coul
22、d promotethe angiogenesis of ischemic myocardium and lessen the injury or necrosis induced byischemiaThe aims of the study were to explore：if Apelin could take part in and protectmyocardial ischemia injury；if Apelin given exogenously could promote proliferation，migration and angiogenesis of endothel
23、iocytes；if Apelin given exogenously couldenchance angiogenesis of ischemic hearts and on which pathway Apelin promoteangiogenesis in order to find an effective way to save ischemic and dying myocardiumObiective1To observe if Apelin could protect ischimic heart by setting up a ratmyocardium ischemic
24、model；2To confirm if Apelin given exogenously could promote heart collateralcirculation and reduce the injury of myocardil ischemia and myocardial infarction；3To study the effects of Apelin on the proliferation，migration and tubeformationof HUVECs；To explore the possible mechanisms of Apelin promoti
25、ng angiopoiesisand if these mechanisms have relations to Apelin and mTORMethods1To detect the protcetion effects of Apelin on ischimic rat(1 1 To ligate the male SD rats left anterior descending(LAD)coronary artery andset up a myrocardial ischimia model Apelin was given(intraperitonealinjection，1pgk
26、gd2)to examine heart rate(HR)，left ventricular end-diastolicpressure(LVEDP)，left ventricular endsystolic pressure(LVESP)and maximal leftventricle developed pressure(LV士dpdtmax)with a cardiac catheterization in order toevalue the effects of Apelin on heart function6(2)Apelin was given(intraperitoneal
27、 injection，1 i,tgkgdx2)to detect the contentof LDH by ligating the male SD ratsLAD and a ischemic model2To examine the effects of Apelin on the collateral circulation of ischemic rathearts(1)To ligate the male SD ratsleft anterior descending(LAD)coronary artery andset up a myocardial ischimia modelA
28、pelin was given fintraperitoneal injection，l 1tgkgd7)to observe the effects ofApelin on collateral circulation in ischemic rats(2)To detect the content of CD3 1 in the myocardium of ischemic rats withenzyme linked immunosorbent assay(ELISA)(3)To dye with AzoBlue and 1vrC and to calculate the myocard
29、ial infarction aerawith a image analysis system(IAS)(4)To observe the angiogenesis ischemic of myocardium dyed with CD3 1immunohistochemistry under a microscope(5)To observe the injury of ischemic myocardium dyed with HE under amicroscope(6)To oberve the protein expression of VEGF and CD3 1 with wes
30、tem blot3To examine the effects of Apelin on the proliferation，migration andangiopoiesis of HUVECs(1)To observe the effect of Apelin on the proliferation of HUVECs by methylthiazolyl tetrazolium(MTT)(2)To explore the effect of Apelin on the migration of HUVECs with Transwell(3)To study the effect of
31、 Apelin on the angiopoiesis of HUVECs withtubeformationResults1The effects of Apelin on heart function of myocardial ischemia in rats Thegroup of Apelin(1 I_tgkgdx2，intraperitoneal injection)enchanced LV士dpdtmax、LVESP and reduced LVEDP significantly compared with other groups at ischemia 48h(P005、00
32、1)2The effects of Apelin on myocardial enzymes of myocardial ischemia in ratsThe Apelin group(1“gkgdx2，intraperitoneal injection)was lower LDH significantlycompared with other groups at ischemia 48h showed by automatic biochemistry7machine(PO01)3Apelin had effects on CD3 1 comem of myocardial ischem
33、ia in rats ELISA ofrat CD3 1 showed that Apelin group(1 ggkgdx7，intraperitoneal injection)was highercompared with other groups(尸005)4The effects of Apelin on myocardial anfarction of myocardial ischemia in ratsThe resuits of infarction aera dyed with AzoBlue and TTC and calculated with IASdiscovered
34、 that Apelin group(1 lxgkgdx7，intraperitoneal injection)was significantlylower compared with other groups伊O05)5The effects of Apelin on MVD of myocardial ischemia in rats TheCD3 1 immunolhistochemistry dye showed that the MVD of Apelin group(1 ggkgdx7，intraperitoneal injection)was significantly high
35、er compared with other groups undermicroscope(尸o0 1)6The effects of Apelin on myocardial construction of myocardial ischemia in ratsThe HE dye showed that the scar of Apelin group(I ggkgdx7，intraperitoneal injection)were significantly much less compared with other groups under microscope7The effects
36、 of Apelin on VEGF and CD3 1 expression of myocardial ischemia inrats The expression of VEGF and CD3 1 were higher significantly compared with othergroups伊O01)8The effects of Apelin on the proliferation of HUVECs The proliferation ofApelin on HUVECs with MTT showed that Apelin group(Apelin 1 umolL)c
37、ouldpromote proliferation of HUVECs significantly compared with other groups(P0O 1)9The effects of Apelin on the migration of HUVECs The migration of Apelin onHUVECs with transwell showed thatApelin group(Apelin 1 ggmL)could promotemigration of HUVECs compared with other groups(P001)1 0The effects o
38、f Apelin on the angiopoiesis of HUVECs The angiopoiesis ofApelin on HUVECs with tubeformation showed thatApelin group(Apelin 60uM)couldpromote angiopoiesis of HUVECs compared with other groups(尸O05)1 1Although Apelin had effects above mentioned the effects，however，the effectsof Apelin on myocardial
39、protection and cardiac function in ischemic rats disapperedwhen Apelin was adminstered with U一73 1 22 simultaneouslyAnd the effects of Apelinon collateral circulation in ischemic rats and proliferation，migration and tubeformation8in HUVECs were disappeared when Apelin was adminstered with rapamycins
40、imultaneouslyConclusions1Apelin could protect cardiac function and injury in ischemic rat hearts2Apelin protected cardiac function and injury in ischemic rat hearts by aclingPLC signal pathway3Apelin could promote myocardium tO expression CD3 1，stimulate angiogenesis，reduce myocardial infarction are
41、a and enchance collateral circulation in ischemicmyocardium4Apelin could facilitate prolifeation，migration and tubeformation in HUVECs5Apelin could promote collateral circulation in ischemic hearts andtubeformation in HUVECs by exciting roTOR receptors and could be blocked by theantagon of mTORrapam
42、ycinKev wordsApelin；myocardial ischemia；CD3 1 protein；VEGF protein；HUVECs；collateralcirculation；signal transduction9英文缩略语英文缩写 英文全称 中文全称APJ angiotensin II Protein J 血管紧张素II相关受体蛋白JATl angiotensin II Xype l 血管紧张素II I型受体GPCRs G protein coupling receptors G蛋白藕连受体P13K phosphaIidylinositol 3-OH kinase 磷脂酰肌
43、醇3激酶Akt serine threonine kinase 丝苏氨酸激酶MVD microvessel density 微血管密度VEGF Vascular Endothelial Growth Factor 血管内皮生长因子mTOR mammalian target ofrapamycin 哺乳动物雷帕霉素靶蛋门DMEM dulbeccoS minimum essential medium 达尔伯克必需基本培养基LDH Lactate dehydrogenase 乳酸脱氢酶ELISA enzyme linked immunosorbent assay 酶联免疫吸附测定HUVECs hum
44、an umbilical vein endothelial cells 人类脐静脉内皮细胞FBS fetal bovine serum 胎牛血清MTT methyl thiazolyl tetrazolium 甲基噻哗基Jq呻测定DMSO dimethyl sulphoxide 二甲基、亚砜FGF fibroblast growth factor 成纤维细胞生长冈子LAD left anterior descending coronary artery 左冠状动脉前降支RAAS reninangiotensinaldosterone system 肾素一血管紧张素-醛嘲酮系统PBS phosp
45、hate buffered solution 磷酸盐缓冲溶液LVESP left ventricular endsystolic pressure 左心室收缩术压LVEDP left ventricular enddiastolic pressure 左心窄舒张未期压IN+dpdtm戕maximal left ventricle developed pressure左室内压最大升降速度N LS nuclear location signal 核定位信号HIV human immunodeficiency virus 人类免疫缺陷病毒MPTP mitochondrial permeability
46、 transition pore心肌细胞线粒体通透性转运孔lOCOPD chronic obstmctire pulmonary disease 慢性阻塞件肺疾病ESRK extracellular signalregulated kinase 细胞外信号调节激酶CHO Chinese h锄ster ovotid 中囡仓鼠卵细胞ANP atri啪n撕uretic peptide 心房利钠肽CD31 plateletendothelial cell adhesion moleculel血小板内皮细胞粘附分子1TNF rumor necrosis factor 肿瘤坏死I大】子PLC phospholipase C 磷脂酶C论文Apelin对心肌缺血大鼠心脏侧枝循环的影响刖 菁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一t要原冈，所以有必要进行积极有效的再灌注治疗，以挽救濒死心肌，减少心肌缺jJL，阽坏死，所以，对缺血心肌进行积极有效的再灌注治疗可以挽救濒死心肌，减少心肌缺血性坏死。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，但是
