【作者】 姜大春；

【导师】 何国祥；

【作者基本信息】 第三军医大学 ， 内科学， 2009， 博士

【摘要】 研究背景:急性心肌梗死(Acute myocardial infarction,AMI)死亡率高,即使幸存者,心功能亦有不同程度的损害,严重危害人们健康。它不仅影响病人的生活质量,而且也给患者家庭和社会增加了过重的负担。近20年来,AMI的诊断和治疗取得了长足进步。一方面,溶栓、经皮冠状动脉介入治疗(Percutaneous Coronary Intervention,PCI)和冠状动脉旁路移植术(Coronary Artery Bypass Grafting,CABG)等血运重建治疗极大地改善了这些病人的预后,另一方面,却又引起了不容忽视的心肌缺血再灌注损伤(Ischemia Reperfusion Injury,IRI),从而降低了其临床疗效。IRI的发生和发展过程是一个多因素相互作用的级联反应,主要的损伤机制有自由基损伤、钙超载、微血管损伤和白细胞的作用等。近年来研究发现,补体、选择素、内皮素和细胞凋亡等均参与了IRI的病理变化过程和结果。可见,IRI可能是多分子、多机制、相互影响、相互促进的病理变化。多年来,IRI一直是心肌缺血治疗中难以解决的问题。虽然尝试了大量的新药和多种防治措施,但由于其作用途径均是外源性的,所以对IRI治疗始终未获得突破性进展,到目前为止仍无系统性治疗措施。在心肌IRI中心肌保护因子的作用一直是研究的热点,血红素氧合酶(Heme Oxygenase,HO)作为一种新型的心肌保护因子,通过其产物的抗炎、抗氧化、抗凋亡和抗心律失常等作用,可在心肌缺血再灌注损伤中发挥重要的保护功能。HO是降解亚铁血红素(Heme)为一氧化碳(CO)、亚铁离子(Fe2+)和胆绿素的胞内限速酶。HO-1是3种HO同功酶中唯一呈诱导性表达的同工酶。HO-1(血红素氧合酶-1)又称HSP32,属于热休克蛋白(Heat Shock Protein,HSP)家族。和其它HSP一样,HO-1的诱导表达是细胞应激时最重要的保护机制之一。HO-1在生理条件下其基因表达处于低水平(除脾脏外),但多种应激因素(如:炎症、缺血、高氧、低氧、或辐射等)可使其表达明显增加。以往的研究表明,HO-1的过度表达,可减轻缺血再灌注损伤,但目前国内外关于HO-1的研究多采用化学诱导剂氯化高铁血红素、钴原卟啉(如:Hemin、CoPP)或抑制剂锌卟啉(如:ZnPP),而化学诱导剂在诱导体内HO-1表达增加的同时可能还会影响体内其他基因的表达,而且有研究显示Hemin本身可引起线粒体的损伤,同时锌卟啉在抑制HO-1的同时也会抑制其他酶(如:NO合酶、鸟苷酸环化酶)的活性,从而影响对HO-1抗缺血再灌注损伤评估。近年来,通过转基因技术将具有细胞保护的基因用于体外转染,逐渐成为组织抗损伤极具希望的策略。而过去对于HO-1转基因技术研究多集中肝、肾移植物的抗缺血再灌注损伤作用方面,其结果令人满意,而对于心肌的缺血再灌注损伤方面研究甚少。这些正是我们构建携带HO-1基因重组腺病毒对大鼠心肌缺血再灌注损伤研究的重要出发点。研究目的和途径:1.Ad-HO-1重组腺病毒载体构建和鉴定;2.将Ad-HO-1重组腺病毒载体感染培养的乳鼠心肌细胞,探讨Ad-HO-1感染靶细胞的效率、目的基因表达产物水平,并通过培养心肌细胞缺氧再复氧模型模拟缺血再灌注模型的建立,从细胞水平探讨HO-1基因对心肌细胞缺氧再复氧损伤的影响及其机制;3.Ad-HO-1重组腺病毒载体预先间接冠脉内转染大鼠心肌后建立心肌缺血再灌注损伤模型,观察Ad-HO-1在体转染对心肌缺血再灌注损伤的保护作用及其机制,为缺血再灌注损伤的防治提供新的途径。研究方法:应用高效的细菌内同源重组方法成功构建重组腺病毒质粒pAdEasy-HO-1,经293细胞包装后,扩增、浓缩,最终获得高效、稳定表达HO-1基因腺病毒Ad-HO-1。采用PCR方法对重组体腺病毒进行鉴定,利用穿梭质粒pAdTrack-CMV中带有GFP报告基因,对病毒滴度和感染效率进行监测。体外实验:采用改良方法分离和培养乳鼠原代心肌细胞,测定重组腺病毒介导的基因转导效率及HO-1表达;建立心肌细胞缺氧、复氧模型模拟心肌缺血再灌注,并进行目的基因转染。观察心肌细胞搏动和形态学变化,采用台盼蓝排斥法检测心肌细胞存活率,测定培养上清液乳酸脱氢酶(LDH)和肌酸磷酸激酶同工酶(CK-MB )含量,丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性,荧光成像系统测定细胞内钙离子浓度。RT-PCR及Western blot法检测心肌细胞HO-1表达,TUNEL法检测心肌细胞凋亡。体内实验:健康雄性SD大鼠随机分为4组:假手术组(SH组),生理盐水组(NS组),腺病毒-荧光蛋白组(Ad组)和腺病毒-HO-1组(HO组)。后3组用自制无损伤血管钳阻断主、肺动脉循环10秒钟,同时于心尖部分别注射单纯生理盐水1ml、含空载体腺病毒(5.0×109PFU)或含重组HO-1腺病毒(7.5×109PFU)的生理盐水1ml,术后3天采用结扎左冠状动脉前降支30min,再灌注120min的方法建立心肌缺血再灌注模型,采用颈动脉插管法测定大鼠缺血再灌注各时相血流动力学指标,再灌注120min后,抽血测定血清心肌酶LDH、CK-MB,并处死大鼠,取左心室缺血部位心肌标本,在荧光显微镜下观察心肌细胞荧光蛋白的表达,计算转染率,TTC法测定左心室心肌梗死面积,并测定心肌组织超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量,电镜下观察心肌细胞的超微结构,RT-PCR、Western blot和TNUEL法检测心肌组织HO-1mRNA、蛋白的表达和心肌细胞的凋亡。研究结果:1.成功构建了重组腺病毒载体Ad-HO-1,经293细胞大量扩增,获得病毒滴度约2×1011pfu/ml;2.心肌细胞转染重组腺病毒Ad-HO-1后12小时至3天均可检测到HO-1mRNA及蛋白的表达,表明HO-1的表达比较稳定;3.细胞实验结果:正常心肌细胞HO-1mRNA表达水平极低(0.093±0.037),与正常对照组(C组)相比,缺氧复氧组(IR组)、空载体组(Ad组)和基因转染实验组(HO组)心肌细胞HO-1mRNA表达(分别为0.337±0.048、0.340±0.082、0.775±0.058)显著升高( P<0.01),与IR组和Ad组相比,HO组HO-1mRNA表达进一步显著增高(P<0.01),各组HO-1蛋白表达与其mRNA表达类似;IR组的细胞存活率降低,基因转染组(HO组)的细胞存活率(87.3±2.6%)显著高于IR组和Ad组(83.0±1.6%、81.6±1.7%)(P<0.01);与对照组(114.3±13.2次/min)相比,IR组和Ad组搏动频率(65.3±5.1次/min、58.9±4.4次/min)显著减慢(P<0.01),HO组搏动频率(97±3.4次/min)显著高于IR组(P<0.01),但显著低于C组(P<0.01);IR组和Ad组LDH活性、CK-MB含量较C组显著升高(P<0.01),HO-1基因转染预处理后LDH活性、CK-MB较IR组和Ad组显著降低(P<0.01),但与C组比差异仍有显著性(P<0.01);IR组和Ad组SOD活性(6.63±1.41、6.75±1.67 U/mg)较C组(12.88±1.46 U/mg)显著下降,HO-1基因转染预处理后SOD活性(9.25±1.04 U/mg)均较IR、Ad组升高(P<0.01),但显著低于C组(P<0.01);细胞内钙离子测定结果显示,IR组和Ad组细胞内游离钙离子([Ca2+]i)离子浓度(483.6±4.5nmol/L、487.3±5.1nmol/L)较正常对照组(183.4±4.9nmol/L)显著升高(P<0.01),HO-1基因转染预处理后[Ca2+]i含量(235.5±10.0 nmol/L)较IR组降低(P<0.01);与C组(3.12±0.83%)比较,IR和Ad组凋亡率显著增加(P<0.01),而HO-1基因转染预处理后心肌细胞凋亡率(13.13±1.72%)明显低于IR组和Ad组(27.88±2.23%、26.38±1.51%)(P<0.01)。IR组和Ad组各指标均无显著差异(P>0.05)。4.在体实验结果:仅Ad和HO组心肌观察到荧光蛋白的表达,两者无显著差异(P>0.05),其转染率分别为54.2±6.3%和56.8±7.0%;SH组见心肌组织极少量HO-1mRNA、蛋白表达。在心肌缺血再灌注后,NS和Ad组心肌组织HO-1mRNA、蛋白表达高于SH组(P<0.01),HO组心肌组织HO-1mRNA、蛋白表达非常显著地高于Ad和NS组(P<0.01);与NS组比较,Ad组HO-l蛋白及HO-lmRNA表达差异无统计学意义(P>0.05);Ad组、NS组和HO组SBP、DBP、MAP、±dp/dtmax绝对值在缺血30min和再灌注1h、2h时相点低于SH组,差异有显著性(P<0.01),HO组SBP、DBP、MAP、±dp/dtmax绝对值均高于相同时相点NS组和Ad组(P<0.01),各时相点HO组的SBP、DBP、MAP、+dp/dtmax和-dp/dtmax等指标恢复率(%)均相应显著高于NS组和Ad组(P<0.01);与SH组相比,NS组、Ad组和HO组血清LDH和CK-MB水平均明显升高(P<0.01),表明心肌组织因缺血再灌注而造成损伤,但HO组血清LDH和CK-MB水平与NS组和Ad组相比显著减少(P<0.01);与NS组和Ad组比较,HO组心肌病理改变减轻,梗死心肌重量(ISW)和梗死面积(ISW/AARW)显著降低(P<0.01);与SH组相比,NS、Ad组和HO组MDA含量均明显升高,SOD活性降低(P<0.01),表明心肌组织因缺血再灌注损伤造成氧自由基的改变,但与NS和Ad组相比,HO组MDA含量明显减少,SOD活性显著升高(P<0.01);SH组仅见极少数散在的凋亡细胞,心肌缺血再灌注后凋亡细胞数明显增加,NS、Ad和HO组心肌细胞凋亡指数显著高于SH组(4.53±1.81)(P<0.01),但与NS、Ad组(26.46±4.23%、25.15±3.11%)比较,HO组心肌细胞凋亡指数(16.84±2.88%)显著下降(P<0.01),NS和Ad组两组心肌细胞凋亡指数差异无统计学意义(P>0.05)。研究结论:1.本实验采用腺病毒AdEasy系统,应用细菌内同源重组方法成功构建重组腺病毒载体Ad-HO-1,经293细胞扩增,获得病毒滴度约2×1011pfu/ml。2.本实验成功建立了体外乳鼠心肌细胞缺氧复氧模型,腺病毒介导的HO-1基因高表达对乳鼠心肌细胞缺氧复氧损伤有显著的保护作用,这一作用与其抑制心肌细胞凋亡、抑制细胞内钙超载、减少氧自由基生成有关。3.本实验建立了在体大鼠心肌缺血再灌注损伤模型,通过腺病毒介导HO-1基因预先间接冠脉内转染致在心肌中高表达HO-1,能够促进再灌注后心功能恢复、减少心肌酶的释放、缩小心肌梗死面积、减轻心肌组织病理损伤,从而产生心肌保护作用。并进一步证实了HO-1抗缺血再灌注心肌损伤作用机制与抗氧化和抑制心肌细胞凋亡有关。4.本研究结果有利于加深对HO-1在心肌缺血再灌注损伤中的作用及其作用机制的认识,HO-1基因转染可望成为心肌缺血再灌注损伤防治的一种新途径和方法。更多还原

【Abstract】 Background:Acute myocardial infarction (AMI) leads to high mortality rate, cardial disfunction, poor quality of life, damage to human health and burden to families and society. During the recent two decades, there is a tremendous development in the diagnosis and therapy of AMI. Percutaneous Coronary Intervention(PCI) and Coronary Artery Bypass Grafting(CABG)improve the prognosis of AMI, however the side-effect such as Ischemia reperfusion injury(IRI) decreases theraputic effect.The occurance and development of IRI are multiple cascade associated with various factors, for example, free radical injury, calcium overload, microvascular injury and the effects of leucocytes. The most recent researches indicate that comlements, selectins, endothelin and apoptosis be involved in the pathophysiological process of IRI. Obviously, IRI is a complex pathological process which multiple factors interact and improve each other. IRI is a troublesome problem of mycardial ischemia for long time, although many new drugs and methods have been used to treat IRI , there is no systemitic procedure to treat IRI since most of the methods are ectogenic. Heme Oxygenase, one of new cardial protective factors, may function in protecting myocardial ischemia through anti-inflamation, anti-oxidation, anti-apoptosis and anti-arhythmia, and is a hot topic of modern research. Heme Oxygenase-1 (HO-1) also known as Heat Shock Protein 32 (HSP32), is the inducible and rate-limiting isoform of heme oxygenase that catalyzes the heme to carbon monoxide, ferrous iron and biliverdin. To date, three identified heme oxygenase isoforms are part of the HO system. The expression of HO-1 is insufficient but is induced by various stress such as inflammation, ischemia, hyperxia, hypoxia and irradiation. Previous studies indicated that the overexpression of HO-1 could release the ischemia reperfusion injury. However, most of the studies used chemical inducers ( Hemin,CoPP) or inhibitors(ZnPP) of HO-1, which inevitably changed the expression of other genes. Futhermore, some reports indicated that hemin might lead to mitochondrail injury, and ZnPP might inhibit other enzyme such as Nitric Oxide synthase or guanylate cyclase. Recently, transgenic technology are used widely to transfect the protective genes to cells or animals, and become more potent strategy to treat tissue injury. Transgenic techniques of HO-1 were mainly focused on liver or kidney transplantation, and the outcomes were good. Since few research on myocardial IRI were used HO-1 transgenic method, in this paper we aim to construct recombinant adenovirus-HO-1 and to explore its role on myocardial IRI in rats.Objectives:1. To construct and identify the recombinant Ad-HO-1 vector.2. Recombinant Ad-HO-1 were used to infect the primary cultured neonate rat myocardial cells, the efficiency and expression of target gene were investigated. By using the model of hypoxia-reoxygen of primary culture of neonate myocardial cells, the effects of HO-1 on cellular hypoxia and re-oxygen were investigated in vitro.3. Ad-HO-1 recombinant was injected into coronary before the IRI rats models were constructed. The effects and mechanism of Ad-HO-1 transfection on myocardial IRI were investigated in vivo to supply new approach to IRI.Methods:By using homologouis recombination techniques the recombinant pAdeasy-HO-1 plasmid was constructed successfully, the recombinant was packaged, concentrated and amplified by 293 cells to get high efficient Ad-HO-1. The recombinant virus was identified with PCR, and the fluorecent protein GFP observation was used to evaluate the efficency of tranfection. In vitro: Neonate myocardial cells were isolated and cultured, and transfection efficency and expression of Ad-HO-1 were investiaged. Myocardial cells hypoxia and re-oxygen model was constructed to be transfected with Ad-HO-1. Morphology and pulsation of myocardial cells were observed, and the cellular survival rate was recorded by the method of trypan blue rejection, apoptosis rate was evaluated by TUNEL method. LDH, CK-MB, MDA and SOD in the supernatant were assayed. The intracellular Ca2+ concentration was examined by fluorecent imaging system. Expression of HO-1 was detected by RT-PCR and western blot. In vivo: Randomized grouping of 48 healthy male SD rats as following:Sham operation(SH), natrual saline(NS), adenovirus vector transfecion (Ad) and Ad-HO-1 transfection(Ad-HO-1), each group included 12 rats. The rats of posterior three groups were clamped for 10 seconds by blood vessel forceps in pulmonary artery or aortic root,and then injected into apex of heart by 1ml NS or 1ml NS with adenovirus(5.0×109PFU), or 1ml NS with recombinant Ad-HO-1 (7.5×109 PFU), the incisions were sutured. Three days after transfection, the rats were transferred into IRI models by clamping in ramus descendens anterior arteriae coronariae sinistrae for 30min and reperfusion for 120 min. The rats of SH groups recepted only clamping in ramus descendens anterior arteriae coronariae sinistrae. Carotid artery cannula was used to record cardial functional parameters, the blood of cardial chambers were collected for myocardial zymogram examination, and the left artrium samples were used to determine the area of infarction with TCC method, to detect MDA and SOD. Transfection effeciency was investigated by fluorecent microscope. Ultrastructure was obsereved by electron microscope.The mRNA and protein level of HO-1 were detected by RT-PCR and Western blot. Cellular apoptosis was evaluated by TUNEL method.Results:1. Recombinant Ad-HO-1 was constructed successfully virus with titre of 2×1011pfu/ml was amplified by 293 cells and collected.2. HO-1 mRNA and protein were detected 12h to 3d after transfection indicating that HO-1 overexpressed stably.3. In Vitro experiments: HO-1 mRNA expressed in normal myocardial cells in low level(0.093±0.037), while the HO-1 expression was higher in cells of IR group, Ad group, HO group(0.337±0.048, 0.340±0.082, 0.775±0.058). The HO-1 expression of HO group was higher than that of IR group or Ad group (P<0.01). For HO-1 protein, similar results were obtained. The cellular survival rate of HO group (87.3±2.6%) was significantly higher than that of IR group(83.0±1.6%) and Ad group (81.6±1.7%)(P<0.01).The pulsation rate of IR group (65.3±5.1beat/min) and Ad group (58.9±4.4beat/min) was deceased obviously compared with that of C group (114.3±13.2beat/min). The pulstaion rate of HO-1 group (97±3.4beat/min) was higher than that of IR group (P<0.01). The bioactivity of LDH and CK-MB of IR group and Ad group was increased significantly than that of C group. The bioactivity of LDH and CK-MB of HO-1 transfection group was less than that of IR group and Ad group. The bioactivity of SOD of IR group(6.63±1.41U/mg) and Ad group(6.75±1.67U/mg ) was decreased significantly than that of C group(12.88±1.46U/mg). The bioactivity of SOD of HO-1 group (9.25±1.04U/mg) was increased significantly than that of IR group (p<0.01). The introcellular calcium of IR group and Ad group (483.6±4.5nmol/L, 487.3±5.1nmol/L) was higher than that of C group (183.4±4.9nmol/L) (P<0.01). The introcellular calcium of HO-1 group (235.5±10.0 nmol/L) was deceased obviously than that of IR group. The apoptosis rate of IR group and Ad group (27.88±2.23%, 26.38±1.51%) was higher than that of C group (3.12±0.83%). The apoptosis rate of HO-1 group (13.13±1.72%) was decreased than that of IR group and Ad group.4. In vivo: By fluorecent microscope, samples of Ad group and HO group were detected GFP positive with transfection rate of 54.2±6.3% and 56.8±7.0%. The expression of HO-1 mRNA and protein in samples of SH group were nearly none. After IRI, HO-1 mRNA and protein of NS group and Ad group were higher than those of SH group (p<0.01). The expression of HO-1 mRNA and protein in HO group were significantly higher than those of NS group and Ad group. There was no statistical difference between NS group and Ad group (P>0.05). Absolute values of SBP, DBP, MAP and±dp/dtmax of SH group after 30min ischimia or reperfusion for 1h, 2h were higher than those of other three group (P<0.01). The aboslute values of SBP, DBP, MAP and±dp/dtmax of HO group were higher than those of NS group and Ad group (P<0.01). The parameters of SBP, DBP, MAP,±dp/dtmax in Ad group, NS group and Ad-HO group were obviously decreased than those of SH group, indicating the success of model construction. Meanwhile, the recovery rate of above parameters in Ad-HO groups were sigificantly higher than those of Ad group and NS group (P<0.01). Tissue samples of HO groups showed less pathological changes, less ISW and ISW/AARW(P<0.01). MDA of SH group was higher than that of Ad group, NS group and HO group (P<0.01), the bioactivity of SOD of SH group was decreased than that of Ad group, NS group and HO group (P<0.01), indicating that free radical changing caused by IRI. While MDA of HO group were less than that of Ad group, NS group (P<0.01), the bioactivity of SOD of HO group was increased than that of Ad group, NS group and HO group (P<0.01). Apoptosis cells detected in SH group (4.53±1.81%) were significantly less than those in other three groups (P<0.01). Apoptosis cells of HO group (16.84±2.88%) were less than those of NS and Ad group (26.46±4.23, 25.15±3.11%) (P<0.01). There was no significant difference between NS and Ad groups (P>0.05). Conclusion:1. By using homologouis recombination techniques the recombinant pAdeasy-HO-1 plasmid was constructed and amplified in 293 cells.2. Myocardial cells hypoxia and re-oxygen model was constructed, the HO-1 overexpression had protective effect on neonate myocardial cells under IRI. The mechanism might be suppressin of apoptosis, inhibiting overload of intercellular calcium and decreasing the release of free radical.3. HO-1 gene was effectively transfected into IRI model rats mediated by adenovirus injection.HO-1 transfection had significant protective effects on Ischemia Reperfusion Injury of myocardium in rats due to restoration of heart function, decreasing the release of marker enzymes, manification of infarction area, remission of pathological injury. The results confirmed that the anti-IRI role of HO-1 associate with decreasing release of free radical, improving anti-oxidase system, lessening lipid peroxidation and inhibiting cell apoptosis.4. Our studies potentialized the role and mechanism of HO-1 on IRI. HO-1 transfection was a potent strategy and method to control IRI of myocardium.更多还原