HO-1基因转染对大鼠心肌缺血再灌注损伤保护作用的实验研究
|
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。
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.
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。
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.
引文
1.第九届全国心血管病学术会议纪要。中华心血管病杂志,2006;38(8):67-69。
2. Carden DL, Granger DN. Pathophysiology of ischaemia-reperfusion injury. Am J Pathol. 2000;190: 255-266.
3. Mark CA, Patel SR, Schwarz EA, et al. Biologic bypass with the use of adenovirus-mediated gene transfer of the complementary deoxyribonucleic acid for vascular endothelial growth factor 121 improves myocardial perfusion and function in the ischemic porcine heart. J Thorac Cardiovasc Surg,1998,115:168-177.
4. Brien ER, Alpers CE, Stewart DK, et al. Proliferation in primary and restenotic coronary atherectomy tissue:Implication for antiproliferative therapy. Circ Res,1993, 73:223-231.
5. Pickering JG, Weir L, Jekanowsky J, et al. Proliferative activity in peripheral and coronary atherosclerotic plaque among patients undergoing percutaneous revascularization. J Clin Invest,1993,91:1469-1480.
6. Prentice H, Bishopric NH, Hick RN, et al. Regulated expression of a foreign gene targeted to ischemic myocardium. Cardiovasc Res, 1997,35: 567-574.
7. Miller DG, Adam MA, Miller AD,et al. Gene transfer by retrovirus vector occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol, 1990,10:4239-4242.
8. Brody Sl, Crystal RG, Boris-Lawrie K, et al. Adenovirus-mediated in vivo gene transfer. Ann NY Acad Sci,1997,716:90-103.
9. Flugelman MY, Jaklitsch MT, Newman KD, et al. Low level in vivo gene transfer into the arterial wall through a perforated balloon catheter. Circ, 1992,85:1110-1117.
10. Riessen R, Rahimizadeh H,T akeshita S, et al. Successful vascular gene transfer using a hydrogel coated balloon angioplasty catheter. Hum Gene Ther, 1993,4:749-758.
11. Lemarchand P, Jones M, Yamada I, et al. In vivo gene transfer and expression in normal uninjured blood vessels using replication-deficient recombinant adenovirus vectors. Circ Res, 1993,72:1132-1138.
12. Sommer B, Kuznetsov SA, Robey PG, et al. Efficient gene transfer into normal human skeletal cells using recombinant adenovirus and conjugated adenovirus-DNA complex. Calcif Tissue Int. 1999,64:45-51.
13. Ryter SW, Alam J and Choi AM. Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications.Physiol Rev 2006,86: 583-650.
14. Yoshida T, Maulik N, Ho YS, et al. H(mox-1) constitutes an adaptive response to effect antioxidant cardioprotection: a study with transgenic mice heterozygous for targeted disruption of the heme oxygenase-1 gene. Circulation. 2001;103: 1695-1701.
15. Clark JE, Foresti R, Sarathchandra P, et al. Heme oxygenase-1–derived bilirubin ameliorates postischemic myocardial dysfunction. Am J Physiol. 2000; 278: H643-H651.
16. WagenerF A,VolkH D,W illisD, et al. Different faces of the heme oxygenase system in inflammation[J]. Pharmacol Rev.2003;55(3): 551-57.
17. Hangaishi M, Ishizaka N, Aizawa T, et al. Induction of heme oxygenase-1 can act protectively against cardiac ischemia/reperfusion in vivo. Biochem Biophys Res Commun. 2000; 279: 582-577.
18. Soares M P,Lin Y,Anrather J,et al. Expression of heme oxygenase-1 can determine cardiac xenograft survival[J]. Nat Med. 1998; 4(9): 1073-1077.
19. VulapalliSR,Chen Z,Chua BH,etal.Cardioselective over expression of HO-1 prevents I/R-induced cardiac dysfunction and apoptosis. Am J Physiol HeartCirc Physiol.2002;283(2):H688-H694.
20. Annegret B, Elena K, Thomas U. AngiotensinII type 2 receptors: signalling and pathophysiological role. Curr Opin Ephrol and Hyper. 2001;10:239-46.
21. Hart DN,Newton MR,Reece-Smith H,et al.Major histocompatibility complex antigens in the rat pancreas,isolated pancreatic islets,thyroid,and adrenal.Transplantation. 1983,36(4):431-435.
22. Ritter T, Lehmann M, Volk HD. Improvements in gene therapy: averting the immune response to adenoviral vectors.Bio Drugs. 2002;16(1):3-10.
23. Suzuki M, Matsuse T, Isigatsubo Y. Gene therapy for lung diseases: development in the vector biology and novel concepts for gene therapy applications.Curr Mol Med. 2001 ;1(1):67-79.
24. He TC, Zhou S, Costa da L,et al. A simplified system for generating recombinat adenoviruses. Proc Natl Acad Sci U S A.1998;95(5):2509-14.
25.黄培堂等编译。《分子克隆实验指南》第三版,科学出版社,北京,2002年。
26.卢圣栋主编。《现代分子生物学实验技术》高等教育出版社,北京,1993年。
27.姚志建,沈倍奋等编译。《分子生物学实验技术》。科学出版社,北京,1990年。
28.王重庆,李云兰等主编。《高级生物化学实验教程》北京大学出版社,北京,1994年。
29.胡福泉主编。《现代基因操作技术》。人民军医出版社,北京,2000年。
30.李永明,赵玉琪等主编。《实验分子生物学方法手册》。科学出版社,北京,1998年。
31.姜泊,张亚历等主编。《分子生物学常用实验方法》。人民军医出版社,北京,1996年。
32. Zeng M,Smith SK,Siegel F,et al.AdEasy system made easier by lecting the viral backbone plasmid preceding homologous recombination. Biotechniques.2001;31(2):260-62.
33.鄂征主编。《组织培养技术》。人民卫生出版社,北京,1986年。
34.司徒镇强,吴军正主编。《细胞培养》。世界图书出版公司,北京,1996年。
35. Kirshenbaum LA, MacLellan WR, Mazur W,et al. Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus. J. Clin Invest.1993 92: 381-387.
36. Perrella MA, Yet SF. Role of heme oxygenase-1 in cardiovascular function. Curr Pharm Des,2003,9:2479-248.
37. Naglaa KI, Andrew D, Gregory YH, et al. Hemoxygenase-1 in cardiovascular disease. J Am Coll Cardiol, 2008, 52: 971-978.
38. Cao J, Inoue K, Li X, Drummond G ,et al. Physiological significance of heme oxygenase in hypertension. Inter J biochemistry & cell biology, 2009,41:1025-33.
39. Joseph FN, Ashok JD. Upregulating the heme oxygenase system suppresses left ventricular hypertrophy in adult spontaneously hypertensive rats for 3 months.J Cardiac Fail, 2009,12:1-13.
40. Tang, YL et al. Protection from ischemic heart injury by a vigilant heme oxygenase-1 plasmid system. Hypertension, 2004, 43:746-751.
41. Muller, OJ et al. Improved cardiac gene transfer by transcriptional and transductional targeting ofadeno-associated viral vectors.Cardiovascular Research, 2006,70:70-78.
42. LI jianjun, LI Gengshan, Huang Congxin,et al.Comparative study of catheter-mediated gene transfer into heart.Chin Med J, 2002, 115:612-613.
43. Masamichi K, Dean M. Anselmo, RW,et al. A novel strategy against ischemia and reperfusion injury: cytoprotection with heme oxygenase system. Transplant Imm 2002,9:227-233
44. Barr E, Carroll J, Kalynych, A.M,et al. Efficient catheter-mediated gene transfer into the heart using replication-defective adenovirus. Gene Ther, 1994,1: 51-58.
45. Luo D. A new solution for improving gene delivery. Trends Biotechnol. 2004;22(3): 101-3.
46. Read ML, Logan A. 11th annual congress of the European Society of Gene Therapy. Exper Rev Antican Ther. 2004;4(1):49-51.
47. Grill J, Geoerger B, Lamfers M, et al. Conditionally replicative adenoviruses: a second wind for cancer gene therapy. Bull Cancer. 2003;90(12):1039-48.
48. Baum BJ, Goldsmith CM, Kok MR, et al. Advances in vector-mediated gene transfer. Immunol Lett. 2003;90(2-3):145-9.
49. Fukuhara H, Hayashi Y, Yamamoto N, et al. Improvement of transduction efficiency of recombinant adenovirus vector conjugated with cationic liposome for human oral squamous cell carcinoma cell lines.Oral Oncol. 2003 ;39(6):601-9.
50. Hedman M, Hartikainen J, Syvanne M,et al. Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia: phase II results of the Kuopio Angiogenesis Trial (KAT).Circulation. 2003 ;107(21):2677-83.
51. Hubberstey AV, Pavliv M, Parks RJ. Cancer therapy utilizing an adenoviral vector expressing only E1A.Cancer Gene Ther. 2002 ;9(4):321-9.
52. Danos O, Mullligan RC. Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc Natl Acad Sci USA. 1988;85:6460-64.
53. Mulligan RC. The basic science of gene therapy. Science. 1993;260:926-932.
54. Miller DG, Adam MA, Miller AD. Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol. 1990;10: 4239-42.
55. Friedman T, Yee JK. Pseudotyped retroviral vectors for studies of human gene therapy. Nature Med. 1995;1: 275-77.
56. Shashikant CS, Ruddle FH. Impact of transgenic technologies on functional genomics.Curr Issues Mol Biol. 2003;5(3):75-98.
57. Grimes BR, Warburton PE, Farr CJ. Chromosome engineering: prospects for gene therapy. Gene Ther. 2002;9(11):713-8.
58. Wong LH, Saffery R, Choo KH. Construction of neocentromere-based human minichromosomes for gene delivery and centromere studies.Gene Ther. 2002 ; 9(11):724-6.
59. Saffery R, Choo KH. Strategies for engineering human chromosomes with therapeutic potential.J Gene Med. 2002 ;4(1):5-13.
60. Park J,Ries J,Gelse J,et al.Bone regeneration in critical sized effects by cell mediated BMP2 gene transfer:acomparison of adenoviral vectors and liposomes.Gene Ther. 2003,10(13):1089 -98.
61. Ramesh R,Saeki T,Templeton NS,et al.Successful treatment of primary and disseminated human lung cancers by systemic delivery tumour suppressor genes using an improved liposome vector.Mol Ther. 2001,3(3):337-50.
62. MizuguchiH,KayMA.Efficient construction of a recombinant adenovirus vector by an improved in vitro ligation method.Hum Gene Ther.1998;9(17):2577-83.
63. Mark CA, Patel SR, Schwarz EA, et al. Biologic bypass with the use of adenovirus-mediated gene transfer of the complementary deoxyribonucleic acid for vascular endothelial growth factor 121 improves myocardial perfusion and function in the ischemic porcine heart. J Thorac Cardiovasc Surg,1998,115:168-177.
64. Brien ER, Alpers CE, Stewart DK, et al. Proliferation in primary and restenotic coronary atherectomy tissue:Implication for antiproliferative therapy. Circ Res,1993,73:223-231.
65. Pickering JG, Weir L, Jekanowsky J, et al. Proliferative activity in peripheral and coronary atherosclerotic plaque among patients undergoing percutaneous revascularization. J Clin Invest,1993,91:1469-1480.
66. Prentice H, Bishopric NH, Hick RN, et al. Regulated expression of a foreign gene targeted to ischemic myocardium. Cardiovasc Res, 1997,35: 567-574.
67. Miller DG, Adam MA, Miller AD,et al. Gene transfer by retrovirus vector occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol, 1990,10:4239-4242.
68. Brody Sl, Crystal RG, Boris-Lawrie K, et al. Adenovirus-mediated in vivo gene transfer. Ann NY Acad Sci,1997,716:90-103.
69. Flugelman MY, Jaklitsch MT, Newman KD, et al. Low level in vivo gene transfer into the arterial wall through a perforated balloon catheter. Circ, 1992,85:1110-1117.
70. Riessen R, Rahimizadeh H,T akeshita S, et al. Successful vascular gene transfer using a hydrogel coated balloon angioplasty catheter. Hum Gene Ther, 1993,4:749-758.
71. Lemarchand P, Jones M, Yamada I, et al. In vivo gene transfer and expression in normal uninjured blood vessels using replication-deficient recombinant adenovirus vectors. Circ Res, 1993,72:1132-1138.
72. Sommer B, Kuznetsov SA, Robey PG, et al. Efficient gene transfer into normal human skeletal cells using recombinant adenovirus and conjugated adenovirus-DNA complex. Calcif Tissue Int. 1999,64:45-51.
73. Wilson JM. Adenoviruses as gene delivery vehicles. N Engl J Med. 1996;334: 1185-1187.
74. Harary L, Farley B. In vitro studies of single isolated beating heart cells.Science, 1960, 131:16741675.
75. Simpson P, Savion S.Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cells.Cross-striations, ultrastructure,and chronotropic response to isoproterenol.Circ Res,1982,50:101-116.
76. Komuro I, Kaida T,Shibazaki Y,et al.Stretching cardiac myocytes stimulates protooncogene expression.J Biol Chem,1990,265:3595-3598.
77.司徒镇强,吴军正主编。《细胞培养》第一版。西安,世界图书出版公司,2004,250-252。
78.蔡文琴主编。《现代实用细胞与分子生物学实验技术》。人民军医出版社,北京,2003年。
79.颜子颖,王海林编译。《精编分子生物学指南》。科学出版社,北京,1998。
80.黄培堂,俞炜源,陈添弥等译。《PCR技术实验指南》。科学出版社,北京,1999年。
81. Ferrier GR,Moffat MP,Lukas A.Possible mechanisms of ventrieular arrhythmiaselieited by ischemia followed by reperfusion.Cire Res.1985:56:184.
82. Koyama T,Temma K,Akera T.Reperfusion-induced contracture develops with a decreasing [Ca2+]:in single heart cells.Am J physiol.1991,261:Hlll5.
83. Chen XW, Zhang XY, Kubo H, etal. Ca2+ infux- induced sarcoplasmic reticulum Ca2+ overload causes mitochondrial-dependent apoptosis inventricular myoeytes.Circ Res,2005,97(11):1009-1017.
84. Altavilla D,Dendato B,Campo GM,etal.IRFI042,a novel dual vitamin E-like antioxidant,inhibits activation of nuclear factor-KB and reduces the inflammatory response in myocardial ischemia-reperfusion injury.Cardiovasc Res, 2000, 47(3): 515-528,
85. Thandroyen FT, Bellotto D,Katayama A,et al.Subcellular electrolyte alterations during progressive hypoxia following reoxygenation in isolated neonatal rat ventricular myocytes.Cire Res,1992,71(l):106-112.
86. Kuramochi T,Honda M,Tanaka K,et al.Contrasting effects of an angiotensin converting enzyme inhibitor and a calcium antagonist on calcium transients in isolated rat cardiac myocytes.Cardiovasc Res,1994;28:1407-1413.
87. Folkman J.Tumor angiogenesis:therapeutic implications.N Engl J Med.1971;285: 1182–1186.
88. Okamoto N,Tobe T,Hackett SF,Ozaki H,et al.Animal model:transgenic mice with increased expression of vascular endothelial growth factor in the retina:a new model of intraretinal and subretinal neovascularization.Am J Pathol.1997;151:281-291.
89. Baffi J,Byrnes G,Chan CC, et al.Choroidal neovascularization in the rat induced by adenovirus mediated expression of vascular endothelial growth factor.Invest Ophthalmol Vis Sci.2000;41:3582-3589.
90. Frank RN,Amin RH,Eliott D,Puklin JE,Abrams GW.Basic fibroblast growth factor and vascular endothelial growth factor are present inepiretinal and choroidal neovascular membranes.Am J Ophthalmol.1996;122:393-403.
91. Hamm CW,Katus HA.New biochemical markers for myocardial cell injury.Curr Opin Cardiol,1995;10(4):355-360.
92. Hangaishi M,Ishizaka N,Aizawa T,et al.Induction of heme oxygenase-1 can act protectively against cardiac ischemia reperufusion in vivo.Bioehem Biophys ResCommun,2000:279:582-588.
93. Clark , JE,Foresti R,Sarathchandra P, et al.Heme oxygenase-1 derived bilirubin ameliorates postischemic myocardial dysfunetion.Am J Physio1 Heart Circ Physio1,2000;278:643-651.
94. Gottlieb RA,Burleson KO,Kloner RA,et al.Reperfusion injury induces apoptosis in rabbit cardiomyocytes.J Clin Invest,1994;94(4):1621-1628.
95. Henry F,Deborah G.Apoptosis in ischemic and reperfusion rat myocardium.Circ Res,1996;79(5):949-956.
96. Brown WM,Hotsley WS,Gott JP,et al.Myocardial protection for coronary artery in patients with acute myocardial infarction:resuscitation of ischemic myocardium.Semin in Thorac and Cardiovasc Surg,1995;7(4):191-197.
97. Sharber J.Oxygen radicals and calcium ion flux:role in reperfusion injury following AMI.Dimens Crit Care Nurs,1994;13(5):256-261.
98. Flitter WD.Free radicals and myocardial reperfusion injury.Br Med Bull,1993; 49(3):545-555.
99. Kirshenbaum LA,Singal PK.Changes in antioxide enzyme in isolated cardiac myocyte subjected to hypoxia-reoxygenation.Lab Invest,1992,67(6):796-803.
100. Xie Y,Zhu WZ,Zhu Y,et al.Intermittent high altitude hypoxia protects the heart against lethal Ca2+ overload injury.Life Science,2004; 76(5):559-572.
101. Petrosillog, Ruggierofm, Divenosan, et al.Decreased complex III activity in mitochondria isolated from rat heart subjected to ischemia and reperfusion: role of reactive oxygen species and cardiolipin . FASEB J,2003, 17 (6): 714-716.
102. Goldhaber JI, Qayyum MS. Oxygen free radicals and excitation-contraction coupling . Antioxid Redox Signal, 2000;2 (1): 55-64.
103. Xu B,Dong GH,Liu H, et al.Recombinant human erythropoietin pretreatment attenuates myocardial infarct size:a possible mechanism involves heat shock protein 70 and attenuation of nuclear factor-kappa B.Ann Clin Lab Sci,2005;35(2):161-168.
104. Chandrasekar B,Smith JB,Freeman GL.Ischemia-reperfusion of rat myocardium activates nuclear factor-KB and induces neutrophil infiltration via lipopolysaccharide-induced CXC chemokine.Circulation,2001;103(18):2296-2302.
105. Date T,Belanger AJ,Mochizuki S.Adenovirus-mediated expression of P35 preventshypoxia reoxygenation injury by redueing reactive oxygen species and caspase activity.Cardiovasc Res,2002;55(2):309-319.
106.方喜业,《医学实验动物学》。北京:人民卫生出版社,1995,291-295。
107. Wright MJ,Wightman LML,Latchman DS,et al.In vivo myocardial gene transfer:optimization and evaluation of intracoronaru gene delivery in vivo[J].Gene Therapy,2001;8:1833-1839.
108. FlissH,GattingerD.Apoptosis in ischemic and reperfused rat myocardium.Circ Res,1996;79(5):949-956.
109. Brounwald主编,陈灏珠主译,《心脏病学》第五版。人民卫生出版社,2001。
110.金惠铭主编,《病理生理学》第五版。人民卫生出版社,2000年,152-160。
111. Murry C,Jennings R,Reimer K. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986; 74: 1124-1136.
112. Li J-J,Ueno H,Pan Y,et al.Percutaneous transluminal gene transfer into canine myocardium in vivo by replication-defective adenovirus. Cardiovasc Res.1995;30: 97-105.
113. Ferry N,Heard J. Liver-directed gene transfer vectors. Human Gene Therapy.1998; 9: 1975-1978.
114. Nabel EG,Pompili VJ,Plautz GE,et al. Gene transfer and vascular disease. Cardiovasc Res.1994;28: 445-454.
115. Qin L,Chavin KD,Ding Y,et al.Multiple vectors effectively achieve gene transfer in a murine cardiac transplantation model.Immunosuppression with TGF-1 or vIL-10.Transplantation.1995;59(6):809-816.
116. Donehue JK, Kikkawa K,Tomas AD,et al. Acceleration of widespread adenoviral gene transfer to intact rabbit hearts by coronary perfusion with low calcium and serotonin. Gene Ther.1998;5:630-634.
117. Davidson MJ,Jones JM,Emani S,et al. Cardiac gene delivery with cardiopulmonary bypass.Circulation.2001;104:131-133.
118. Katori M,Buelow R,Ke B,et al.Heme oxygenase-1 overexpression protects rat hearts from cold ischemia reperfusion injury via an antiapoptotic pathway.Transplantation. 2002;73(2): 287-292.
119. Katori M, Busuttil RW, Kupiec-Weglinski J. Heme oxygenase-1 System in organtransplantation. Transplantation, 2002, 74: 905-912.
120. Alok SP, Luis G. Melo, et al. Chronic recurrent myocardial ischemic injury is significantly attenuated by pre-emptive adeno-associated virus heme oxygenase-1 gene delivery. J Am Coll Cardiol 2006;47:635- 43.
121. Yoshida T, Maulik N, Ho YS, Alam J, Das DK. H(mox-1) constitutes an adaptive response to effect antioxidant cardioprotection: a study with transgenic mice heterozygous for targeted disruption of the Heme oxygenase-1 gene. Circulation. 2001;103:1695–701.
122. Becker LB. New concepts in reactive oxygen species and cardiovascular reperfusion physiology. Cardiovasc Res.2004;61:461-70.
123. Sharma HS,Das DK,Verdouw PD. Enhanced expression and localization of heme oxygenase-1 during recovery phase of porcine stunned myocardium. Mol Cell Biochem.1999;196:133-139.
124. Lee TS,Chan LY. Heme oxygenase-1 mediates the anti-inflammatory effect of interleukin-10 in mice. Nat Med.2002;8:240-246.
125. Choi AM.Heme oxygenase-1 protects the heart[J].Circ Res.2001;89:168-173.
126. Maines MD,Panahian N.The heme oxygenase system and cellular defense mechanisms. Do HO-1 and HO-2 have different function? Adv Exp Med Biol. 2001;502:249-272.
127. Hori R,Kashiba M,Toma T,et al.Gene transfection of H25A mutant heme oxygenase-1 protects cells against hydroperoxide-induced cytotoxicity. J Biol Chem.2002;277: 10712-10718.
128. Frangogiannis NG,Smith CW,Entman ML.The inflammatory response in myocardial infarction.Cardiovasc Res.2002;53:31-47.
129. Sun B,Fan H,Honda T,et al.Activation of NF kappa B and expression of ICAM-1 in ischemic-reperfused canine myocardium.J Mol Cell Cardiol.2001;33:109-119.
130. Vachharajani TJ,Work J,Issekutz AC,et al.Heme oxygenase modulates selectin expression in different regional vascular beds.Am J Physiol Heart Circ Physiol.2000;278:H613-617.
131. Hayashi S,Takamiya R,Yamaguchi T,et al.Induction of heme oxygenase-1 suppresses venular leukocyte adhension elicited by oxidative stress:role of bilirubin generated bythe enzyme.Circ Res.1999;85:663-671.
132. Yet SY,Tian R,Layne MD,et al.Cardiac specific expression of heme oxygenase-1 protects against ischemia and reperfusion injury in transgenic mice.Circ. 2001;89:105-107.
133. Wang R,Wu LY.The chemical modification of K-Ca channels by carbon monoxide in vascular smooth muscle cell.J Biol Chem.1997;272:8222-8226.
134. Peng J,Lu R,Ye F,et al.The heme oxygenase-1 pathway is involved in calcitonin gene-related peptide-mediated delayed cardioprotection induced by monophosphoryl lipid A in rats.Regul Pept.2002;103:1-7.
135. Alok SP, Anthony S, Patricia MD et al. Heme-oxygenase-1-induced protection against hypoxia/reoxygenation is dependent on biliverdin reductase and its interaction with PI3K/Akt pathway. J Mol Cell Cardiol. 2007,43: 580-592.
136. Brouard S,Berberat PO,Tobiasch E,et al.Heme oxygenase-1 derived carbon monoxide requires the activation of transcription factor NF-kappa B to protect endothelial cell from tumor necrosis factor-alpha-mediated apoptosis.J Biol Chem.2002;277: 17950-17961.
137. Zweier JL,Fertmann J,Wei G.Nitric oxide and peroxynitrite in postichemic myocardium.Antioxid Redox Signal.2001;3:11-22.
138. Maines MD,Panahian N.The heme oxygenase system and cellular defense mechanisms. DO HO-1 and HO-2 have different function? Adv Exp Med Biol.2001;502:249-272.
139. Hartsfield CL.Cross talk between carbon monoxide and nitric oxide. Antioxid Redox Signal.2002;4:301-307.
140. Kamilla K, Kevin D. Approaches for cardiovascular gene therapy in animal models. Drug Discovery Today: Disease Models.2006,3:305-311.
1. Otterbein LE, Choi AMK. Heme oxygenase: colors of defense against cellular stress.Am J Physiol,2000,279:L1029-L1037.
2. Motterlini R, Foresti R, Intaglietta M, et al. NO-mediated activation of heme oxygenase: endogenous cytoprotection against oxidative stress to endothelium. Am J Physiol Heart Circ Physiol.1996;270: H107-114.
3. Durante W, Kroll MH, Christodoulides N, et al. Nitric oxide induces heme oxygenase-1 gene expression and carbon monoxide production in vascular smooth muscle cells. Circ Res. 1997; 80:557-564.
4. Tian W, Bonkovasky HL, Shibahara S, et al. Urea and hypertonicity increase expression of heme oxygenase-1 in murine renal medullary cells. Am J Physiol Renal Physiol. 2001; 281: F983-991.
5. Chen YH, Lin SJ, Lin MW, et al. Microsatellite polymorphism in promoter of heme oxygenase-1 gene is associated with susceptibility to coronary artery disease in type 2 diabetic patients. Hum Genet. 2002; 111:1-8.
6. Schillinger M, Exner M, Mlekusch W, et al. Heme oxygenase-1 genotype is a vascular anti-inflammatory factor following balloon angioplasty. J Endovasc Ther. 2002; 9:385-394.
7. Otterbein LE, Bach FH, Alam J, et al. Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat Med. 2000; 6: 422-428.
8. Liu XM., Chapman GB., Peyton KJ, et al. Carbon monoxide inhibits apoptosis in vascular smooth muscle cells. Cardiovasc. Res.2002; 55: 396–405
9. Liu XM, Peyton KJ, Ensenat D, et al. Endoplasmic Reticulum Stress Stimulates Heme Oxygenase-1 Gene Expression in Vascular Smooth Muscle. J Biol Chem. 2005; 280:872-877.
10. McDaid J, Yamashita K, Chora A, et al. Heme oxygenase-1 modulates the allo-immune response by promoting activation-induced cell death of T cells. FASEB J. 2005; 19:458-460.
11. Dasilva JL, Tiefenthaler M, Park E, et al. Tin-mediated heme oxygenase gene activationand cytochrome P450 arachidonate hydroxylase inhibition in spontaneously hypertensive rats. Am J Med Sci.1994;307:173 181.
12. Christou H, Morita T, Hsieh CM, et al. Prevention of hypoxia-induced pulmonary hypertension by enhancement of endogenous heme oxygenase-1 in the rat. Circ Res. 2001; 86:1224–1229.
13. Johnson RA, Lavesa M, Deseyn K, et al. Heme oxygenase substrates acutely lower blood pressure in hypertensive rats. Am Phys Soci. 1996; 271:H132-138.
14. Foresti R, Goatly H, Green CJ, et al. Role of heme oxygenase-1 in hypoxia reoxygenation:requirement of substrate heme to promote cardioprotection.Am J Physiol Heart Circ Physiol.2001;281(5):H1976-984.
15. Wu TW, Wu J, Li RK, et al. Albumin-bound bilirubins protect human ventricular myocytes against oxyradical damage. Biochem Cell Biol. 1991; 69: 683– 688.
16. Balla G, Jacob HS, Balla J, et al.Ferritin:a cytoprotective antioxidant strategem of endothelium. J Biol Chem,1992;267:18148-18153.
17. Kaur H,Hughes MN, Green CJ, et al. Interaction of bilirubin and biliverdin with reactive nitrogen species. FEBS Lett, 2003;543 (1-3):113-119.
18. Mancuso C,Bonsignore A, Stasio ED, et al. Bilirubin and S-nitrosothiols interaction:evidence for a possible role of bilirubin as a scavenger of nitric oxide.Biochem Pharmacol,2003;66 (12):2355-2363.
19. Hayashi S, Takamiya R, Yamaguchi T, et al. Induction of heme oxygenase-1 suppresses venular leukocyte adhesion elicited oxidative stress:role ofbilirubin generated by the enzyme. Circ Res,1999;85:663-671.
20. Wagener F,Silva JL, Farley T, et al. Differential effects of heme oxygenase isoforms on heme mediation of endothelial intracellular adhesion molecule 1 expression. Pharmacol Exp Ther,1999;291:416-423.
21. Otterbein LE, Bach FH, Alam J,et al. Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway.Nat Med, 2000;6 (4):422-428.
22. Katori M, Buelow R, Ke B, et al.Heme oxygenase-1 overexpression protects rat hearts from cold ischemia reperfusion injury via an antiapoptotic pathway.Transplantation, 2002;73 (2): 287-292.
23. Akamatsu Y, Haga M, et al. Heme oxygenase-1derived carbonmonoxide protectshearts from transplant-associated ischemia reperfusion injury.FASEB J, 2004;10. 1096-1099.
24. Vulapalli SR, Chen ZY, Chua BHL,et al. Cardioselective overexpression of HO-l prevents I/R-induced cardiac dysfunction and apoptosis.Am J Physiol Heart Circ Physiol.2002;283 (2):H688-694.
25. Melo LG, Agrawal R, Zhang L, et al. Gene therapy strategy for long-term myocardial protection using adeno-associated virus-mediated delivery of heme oxygenase gene. Circulation. 2002; 105:602–607.
26. Foresti R, Sarathchandra P, Clark JE, et al. Peroxynitrite induces heme oxygenase-1 in vascular endothelial cells: a link to apoptosis. Biochem J, 1999;339(Pt 3):729-736.
1. French BA, Mazur W, Geske RS, et al. Direct in vivo gene transfer into porcine myocardium using replication-deficient adenoviral vectors[J]. Circ, 1994,90:2414-2424.
2. Berkner KL. Expression of heterologous sequences in adenoviral vectors[J]. Curr Top Microbiol Immunol,1992,158:39-66.
3. Fallaux FJ, Kranenburg O, Cramer SJ, et al. Characterization of 911:A new helper cell line for the titration and propagation of early-region-deleted adenoviral vectors[J]. Hum Gene Ther, 1996,7:215-222.
4. Mark CA, Patel SR, Schwarz EA, et al. Biologic bypass with the use of adenovirus-mediated gene transfer of the complementary deoxyribonucleic acid for vascular endothelial growth factor 121 improves myocardial perfusion and function in the ischemic porcine heart[J]. J Thorac Cardiovasc Surg, 1998,115:168-177.
5. O‘Brien ER, Alpers CE, Stewart DK, et al. Proliferation in primary and restenotic coronary atherectomy tissue:Implication for antiproliferative therapy[J]. Circ Res,1993,73:223-231.
6. Pickering JG, Weir L, Jekanowsky J, et al. Proliferative activity in peripheral and coronary atherosclerotic plaque among patients undergoing percutaneous revascularization[J]. J Clin Invest,1993,91:1469-1480.
7. Prentice H, Bishopric NH, Hick RN, et al. Regulated expression of a foreign gene targeted to ischemic myocardium[J]. Cardiovasc Res, 1997,35: 567-574.
8. Miller DG, Adam MA, Miller AD,et al. Gene transfer by retrovirus vector occurs only in cells that are actively replicating at the time of infection[J]. Mol Cell Biol, 1990,10:4239-4242.
9. Brody Sl, Crystal RG, Boris-Lawrie K, et al. Adenovirus-mediated in vivo gene transfer[J]. Ann NY Acad Sci,1997,716:90-103.
10. Flugelman MY, Jaklitsch MT, Newman KD, et al. Low level in vivo gene transfer into the arterial wall through a perforated balloon catheter[J]. Circ, 1992,85:1110-1117.
11. Riessen R, Rahimizadeh H,T akeshita S, et al. Successful vascular gene transfer using a hydrogel coated balloon angioplasty catheter[J]. Hum Gene Ther, 1993,4:749-758.
12. Lemarchand P, Jones M, Yamada I, et al. In vivo gene transfer and expression in normal uninjured blood vessels using replication-deficient recombinant adenovirus vectors[J]. Circ Res, 1993,72:1132-1138.
13. Sommer B, Kuznetsov SA, Robey PG, et al. Efficient gene transfer into normal humanskeletal cells using recombinant adenovirus and conjugated adenovirus-DNA complex[J]. Calcif Tissue Int. 1999,64:45-51.
14. Smith TA, Mehaffey MG, Kayda DB, et al. Adenovirus mediated expression of therapeutic plasma levels of human factorⅨin mice[J]. Nature Genet, 1993,5:397-402.
15. Lee LY, Patel SR, Hackett NR, et al. Focal angiogen therapy using intramyocardial delivery of adenovirus vector coding for vascular endothelial growth factor 121[J]. Ann Thorac Surg,2000,69:14-24.
16. Wang Q, Finer MH. Second-generation adenovirus vectors[J]. Nature Med, 1996,2:714-716.
17. Shean MK, Baskin G, Sullivan D, et al. Immunomodulation and adenoviral gene transfer to lungs of nonhuman primate[J]. Hum Gen Ther,2000,11:1047-1055.
18. Newman KD, DunnPF, Owens JL, et al. Adenovirus-mediated gene transfer into normal rabbit arteries results in prolonged vascular cell activation, inflammation and neointimal hyperplasia[J]. J Clin Invest, 1995,95:2955-2965.
19. Gao GP,Yang Y,Wilson JM.Biology of adenovirus vectors with E1 and E4 deletions for liver directed gene therapy[J]. J Virol,1996,70:8934-8943.
20. Amalfitano A,Hauser MA,Hu H,et al Production and characterization of improved adenovirus vectrors with the E1,E2 and E3 genes deleted[J]. J Virol,1998,72:926-933.
21. Lusky M, Christ M, Rittner K,et al. In vitro and in vivo biology of recombinant adenovirus vectors with E1,E1PE2A,or E1PE4 deleted[J]. J Virol,1998,72:2022-2032.
22. Hu H, Serra D, Amalfitano A. Persistence of an [ E12,polymerase2] adenovirus vector despite transduction of a neoantigen into immune competent mice[J]. Hum Gene Ther,1999, 10:355-364.
23. Morsy MA, Gu MC,Motzel S,et al. An adenoviral vector deleted for all viral coding sequences results an enhancde safety and extended expression of a leptin transgene[J]. Proc Natl Acad Sci USA,1998,95:7866-7871.
24. Kochanek S, Clemens PR, Mitani K, et al. A new adenoviral vector:replacement of all viral coding sequences with 28 kb of DNA independently expressing both full2length dystrophin and beta-galactosidase[J]. Proc Natl Acad Sci USA,1996,93:5731-5736.
25. Lieber A,He CY,Kirillova I,et al. Recombinant adenoviruses with large deletions generated by cre2 mediated excision exhibit different biological properties compared with first generation vectors in vitro and in vivo[J]. J Virol,1996,70:8944-8960.
26. Svensson EC,Marshal DJ,Woodard K,et al. Efficient and stable transduction of cardiomyocytes after intramyocardial injection or intracoronary perfusion with recombinantadeno-associated virus vectors[J]. Circulation, 1999,99: 201-205.
27. Rolling F,Nong Z,Pisvin S,et al.Adeno-associated virus-mediated gene transfer into rat carotid arteries[J]. Gene Ther, 1997,4: 757-761.
28. 281.Felgner PL, Barenholz Y, Behr JP,et al.Nomenclature for synthetic gene delivery systems[J]. Hum.Gene Ther, 1997,8: 511-512.
29. Schwartz LB,Moawad J. Gene therapy for vascular disease[J].Ann Vasc Surg,1997,11:189- 199.
30. Kirshenbaum LA, MacLellan WR, Mazur W,et al. Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus[J].Clin. Invest, 1993,92: 381-387.
31. Kaplan JM, St George JA, Pennington SE, et al. Humoral and cellular immune responses to Ad2/CFTR-2[J].Gene Ther,1996,3( 2):117- 127.
32. Christ M, Lusky M, Stieckel F, et al. Gene therapy with recomhinant adenovirus vectors: evaluation of the host immune response[J].Immunol Lett,1997,57( 1997):19-25.
33. Yap T, Brien TO, Tazelaar HD, Immunosuppression prolongs adenoviral mediated transgene expression in cardiac allograft transplantation[J].Cardiovasc Res,1997,35: 529-535.
34. Dipak K, Richard M, Engelman, et al. Molecular targets of gene therapy[J].Ann Thor Surg,1999, 68(5):1929-1933.
35.杨惠玲等,《高级病理生理学》第一版。北京:科学出版社,1998,10:569 - 594.
36. Donahue JK, Kiklawa K,Johivs D,et al.Ultrarapid,highly efficient viral gene transfer to the heart[J]. Proc Natl Acad Sci, 1997, 94:4664-4668.
37. Donahue JK, Kiklawa K, Johivs D,et al.Acceleration of widespread adenoviral gene transfer to intact rabbit hearts by coronary perfusion with low calcium and serotonin[J]. Gene Therapy, 1998, 95:5251-5256.
38. Damien L, Hatem SN, Heimburger M,et al.How to optimize in vivo gene transfer to cardiac myocytes:mechanical or pharmacological procedures [J]? Human Gene Therapy, 2001, 12:1601-1610.
39. Wright MJ, Wightman LML, Latchman DS,et al.In vivo myocardial gene transfer:optimization and evaluation of intracoronaru gene delivery in vivo[J].Gene Therapy, 2001, 8:1833-1839.
40. Davidson MJ, Jones JM, Emani S,et al.Cardiac gene delivery with cardiopulmonary bypass [J]. Circulation, 2001,104:131-133.
41. Jones JM, Wilson KH, Kock WJ,et al.Adenovital gene transfer to the heart duringcardiopulmonary bypass:effect of myocardial protection technique on transgene expression[J]. Euro J Card-Thor Surg, 2002, 21:847-852.
42. LI jianjun, LI Gengshan, Huang Congxin,et al.Comparative study of catheter-mediated gene transfer into heart[J].Chin Med, J 2002, 115:612-613.
43. Riessen R. and Isner J.M. Prospects for site-specific delivery of pharmacologic and molecular therapies[J].Am. CollCardiol, 1994,23: 1234-1244.
44. Barr E, Carroll J, Kalynych, A.M,et al. Efficient catheter-mediated gene transfer into the heart using replication-defective adenovirus[J]. Gene Ther, 1994,1: 51-58.
45. Tomiyasu K, Oda Y, Nomura M,et al. Direct intra-cardiomuscular transfer of beta2-adrenergic receptor gene augments cardiac output in cardiomyopathic hamsters[J].Gene Ther, 2000,7: 2087-2093.
46. Lamping KG, Rios CD, Chun JA, et al. Intrapericardial administration of adenovirus for gene transfer[J].Am J Physiol, 1997,272: H310-H317 .
47. Neyroud N, Nuss HB, Leppo MK,et al.Gene delivery to cardiac muscle[J].Methods Enzymol., 2002,346: 323-334.
48. Feldman LJ. and Steg G. Optimal techniques for arterial gene transfer[J].Cardiovasc. Res., 1997,35: 391-404.
49. Valen G, Paulsson G, Vage J. Induction of inflammatory mediators during reperfusion of the human heart[J]. Ann Thorac Surg, 2001, 71: 22E232
50. Morishita R, Sugimoto T, Aoki M, et al. In vivo transfection of cis element "decoy" against nuclear factor-kappaB binding site prevents myocardial infarction[J]. Nat. Med., 1997,3: 894-899.
51. Sawa Y, Morishita R, Suzuki K ,et al.novel strategy for myocardialprotection using in vivo transfection of cis element 'decoy' against NF kappaB binding site: evidence for a role of NF kappaB in ischemia reperfusion injury[J]. Circulation, 1997,96: 2-4.
52. Kupatt C, Wichels R, Deiss M,et al. Retroinfusion of NF kappaB decoy oligonucleotide extends cardioprotection achieved by CD18 inhibition in a preclinical study of myocardial ischemia and retroinfusion in pigs[J]. Gene Ther, 2002,9: 518-526.
53. Matsui Y, Inobe M, Okamoto H,et al. Blockade of T cell costimulatory signals using adenovirus vectors prevents both the induction and the progression of experimental autoimmune myocarditis[J]. J. Mol. Cell Cardiol, 2002,34: 279-295.
54. Nakano A, Matsumori A, Kawamoto S, et al. Cytokine gene therapy for myocarditis by in vivo electroporation[J]. Hum. Gene Ther, 2001,12: 1289-1297.
55. Cull VS, Bartlett EJ and James CM. Type I interferon gene therapy protects against cytomegalovirus-induced myocarditis[J]. Immunology, 2002,106: 428-437.
56. Watanabe K, Nakazawa M, Fuse K, et al. Protection against autoimmune myocarditis by gene transfer of interleukin-10 by electroporation[J]. Circulation, 2001,104: 1098-1100
57. Lim BK, Choe SC, Shin JO, et al. Local expression of interleukin-1 receptor antagonist by plasmid DNA improves mortality and decreases myocardial inflammation in experimental coxsackieviral myocarditis[J].Circulation,2002,105: 1278-1281.
58. Bilbao G, Contreras JL, Gomez-Navarro J, et al. Genetic modification of liver grafts with an adenoviral vector encoding the Bcl-2 gene improves organ preservation[J]. Transplantation, 1999,67: 775-783.
59. Chen Z, Chua CC, Ho YS, et al. Overexpression of Bcl-2 attenuates apoptosis and protects against myocardial I/R injury in transgenic mice[J]. Am. J. Physiol. Heart Circ.Physiol, 2001,280: H2313-H2320
60. Kirshenbaum LA and de Moissac D. The bcl-2 gene product prevents programmed cell death of ventricular myocytes[J]. Circulation, 1997,96: 1580-1585.
61. Huang J, Ito Y, Morikawa M, Uchida H, et al. Bcl-xL gene transfer protects the heart against ischemia reperfusion injury[J]. Biochem Biophys Res Commun, 2003,311: 64-70.
62. Sawitzki B, Amersi F, Ritter T, et al. Upregulation of Bag-1 by ex vivo gene transfer protects rat livers from ischemia/reperfusion injury[J]. Hum Gene Ther, 2002,13: 1495-1504.
63. Laugwitz KL, Moretti A, Weig HJ, et al. Blocking caspase-activated apoptosis improves contractility in failing myocardium[J]. Hum. Gene Ther, 2001,12: 2051-2063.
64. Matsui T, Li L, del M, et al. Adenoviral gene transfer of activated phosphatidylinositol 3'-kinase and Akt inhibits apoptosis of hypoxic cardiomyocytes in vitro[J]. Circulation, 1999,100: 2373-2379.
65. Miao W, Luo Z, Kitsis RN, et al. Intracoronary, adenovirus-mediated Akt gene transfer in heart limits infarct size following ischemia-reperfusion injury in vivo[J]. J. Mol. Cell Cardiol, 2000,32: 2397-2402.
66. Lehmann TG., Wheeler M.D, Schoonhoven R, et al. Delivery of Cu/Zn-superoxide dismutase genes with a viral vector minimizes liver injury and improves survival after liver transplantation in the rat[J]. Transplantation, 2000,69: 1051- 1057
67. Abunasra HJ, Smolenski RT, Yap J, et al. Multigene adenoviral therapy for the attenuation of ischemia-reperfusion injury after preservation for cardiac transplantation[J]. J Thorac Cardiovasc Surg, 2003,125: 998-1006.
68. Wink DA, Hanbauer I, Krishna MC. Nitric oxide protects against cellular damage and cytotoxicity from reactive oxygen species[J].Proc Natl Acad Sci USA,1993,90:9813～9817.
69. Johnson G, Tsao PS, Mulloy D, et al. Cardioprotective effects of acidified sodium nitrite in myocardial ischemia with reperfusion[J].J Pharmacol Exp Ther,1990,252(1):35～41。
70. Suda T, Mora BN, D'Ovidio F, et al.In vivo adenovirus-mediated endothelial nitric oxide synthase gene transfer ameliorates lung allograft ischemia-reperfusion injury[J]. J Thorac Cardiovasc Surg, 2000, 119: 297-304.
71. Kim KS, Takeda K, Sethi R, et al. Protection from reoxygenation injury by inhibition of rac1[J]. J. Clin. Invest, 1998 ,101: 1821-1826.
72. Melo LG, Agrawal R, Zhang L,et al. Gene therapy strategy for long-term myocardial protection using adeno-associated virus-mediated delivery of heme oxygenase gene[J]. Circulation, 2002,105: 602-607.
73. Karp G. Cell and molecular biology: concepts and experiments. 2nd [M]. New York:John Wiley & ons Inc,1999, 204.
74. Kelly, S. et al. Gene transfer of HSP72 protects cornu ammonis 1 region of the hippocampus neurons from global ischemia: influence of Bcl-2[J]. Ann. Neurol. 2002,52:160–167.
75. Creagh EM, Carmody RJ, Cotter TG. Heat shock protein 70 inhibits caspase-dependent and -independent apoptosis in Jurkat T cells[J]. Exp Cell Res. 2000,257:58–66.
76. Wong HR, Ryan M Wispe JR. Stress response decreases NF-kappaB nuclear translation and increases 1-appaB alpha expression in A549 cells[J]. J Clin lnvest,1997,99(10):2423-2428
77. Ken Suzuki et al,.in vivo gene transfection with heat shock protein 70 enhances myocardial tolerance to ischemia-reperfusion in jury in rat[J]. J clin invest.1996,99(7),1645-1650
78. Jay jayakumar et al, Transfection of dornor hearts with heat shock protein 70 gene protects cardiac function against ischemia-reperfusion injury[J].Circulation.2000,88: 1264–1272.
79. Jayakumar J, Suzuki K, Sammut IA, et al. Heat shock protein 70 gene transfection protects mitochondrial and ventricular function against ischemia-reperfusion injury[J]. Circulation, 2001,104: 1303-1307.
80. Gripes CL, Watkins MW, Helmer G, et al. Angiogenic gene therapy (AGENT) trial in patients with stable angina pectoris[J]. Circulation,2002 .105:1291-1297.
2. Carden DL, Granger DN. Pathophysiology of ischaemia-reperfusion injury. Am J Pathol. 2000;190: 255-266.
3. Mark CA, Patel SR, Schwarz EA, et al. Biologic bypass with the use of adenovirus-mediated gene transfer of the complementary deoxyribonucleic acid for vascular endothelial growth factor 121 improves myocardial perfusion and function in the ischemic porcine heart. J Thorac Cardiovasc Surg,1998,115:168-177.
4. Brien ER, Alpers CE, Stewart DK, et al. Proliferation in primary and restenotic coronary atherectomy tissue:Implication for antiproliferative therapy. Circ Res,1993, 73:223-231.
5. Pickering JG, Weir L, Jekanowsky J, et al. Proliferative activity in peripheral and coronary atherosclerotic plaque among patients undergoing percutaneous revascularization. J Clin Invest,1993,91:1469-1480.
6. Prentice H, Bishopric NH, Hick RN, et al. Regulated expression of a foreign gene targeted to ischemic myocardium. Cardiovasc Res, 1997,35: 567-574.
7. Miller DG, Adam MA, Miller AD,et al. Gene transfer by retrovirus vector occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol, 1990,10:4239-4242.
8. Brody Sl, Crystal RG, Boris-Lawrie K, et al. Adenovirus-mediated in vivo gene transfer. Ann NY Acad Sci,1997,716:90-103.
9. Flugelman MY, Jaklitsch MT, Newman KD, et al. Low level in vivo gene transfer into the arterial wall through a perforated balloon catheter. Circ, 1992,85:1110-1117.
10. Riessen R, Rahimizadeh H,T akeshita S, et al. Successful vascular gene transfer using a hydrogel coated balloon angioplasty catheter. Hum Gene Ther, 1993,4:749-758.
11. Lemarchand P, Jones M, Yamada I, et al. In vivo gene transfer and expression in normal uninjured blood vessels using replication-deficient recombinant adenovirus vectors. Circ Res, 1993,72:1132-1138.
12. Sommer B, Kuznetsov SA, Robey PG, et al. Efficient gene transfer into normal human skeletal cells using recombinant adenovirus and conjugated adenovirus-DNA complex. Calcif Tissue Int. 1999,64:45-51.
13. Ryter SW, Alam J and Choi AM. Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications.Physiol Rev 2006,86: 583-650.
14. Yoshida T, Maulik N, Ho YS, et al. H(mox-1) constitutes an adaptive response to effect antioxidant cardioprotection: a study with transgenic mice heterozygous for targeted disruption of the heme oxygenase-1 gene. Circulation. 2001;103: 1695-1701.
15. Clark JE, Foresti R, Sarathchandra P, et al. Heme oxygenase-1–derived bilirubin ameliorates postischemic myocardial dysfunction. Am J Physiol. 2000; 278: H643-H651.
16. WagenerF A,VolkH D,W illisD, et al. Different faces of the heme oxygenase system in inflammation[J]. Pharmacol Rev.2003;55(3): 551-57.
17. Hangaishi M, Ishizaka N, Aizawa T, et al. Induction of heme oxygenase-1 can act protectively against cardiac ischemia/reperfusion in vivo. Biochem Biophys Res Commun. 2000; 279: 582-577.
18. Soares M P,Lin Y,Anrather J,et al. Expression of heme oxygenase-1 can determine cardiac xenograft survival[J]. Nat Med. 1998; 4(9): 1073-1077.
19. VulapalliSR,Chen Z,Chua BH,etal.Cardioselective over expression of HO-1 prevents I/R-induced cardiac dysfunction and apoptosis. Am J Physiol HeartCirc Physiol.2002;283(2):H688-H694.
20. Annegret B, Elena K, Thomas U. AngiotensinII type 2 receptors: signalling and pathophysiological role. Curr Opin Ephrol and Hyper. 2001;10:239-46.
21. Hart DN,Newton MR,Reece-Smith H,et al.Major histocompatibility complex antigens in the rat pancreas,isolated pancreatic islets,thyroid,and adrenal.Transplantation. 1983,36(4):431-435.
22. Ritter T, Lehmann M, Volk HD. Improvements in gene therapy: averting the immune response to adenoviral vectors.Bio Drugs. 2002;16(1):3-10.
23. Suzuki M, Matsuse T, Isigatsubo Y. Gene therapy for lung diseases: development in the vector biology and novel concepts for gene therapy applications.Curr Mol Med. 2001 ;1(1):67-79.
24. He TC, Zhou S, Costa da L,et al. A simplified system for generating recombinat adenoviruses. Proc Natl Acad Sci U S A.1998;95(5):2509-14.
25.黄培堂等编译。《分子克隆实验指南》第三版,科学出版社,北京,2002年。
26.卢圣栋主编。《现代分子生物学实验技术》高等教育出版社,北京,1993年。
27.姚志建,沈倍奋等编译。《分子生物学实验技术》。科学出版社,北京,1990年。
28.王重庆,李云兰等主编。《高级生物化学实验教程》北京大学出版社,北京,1994年。
29.胡福泉主编。《现代基因操作技术》。人民军医出版社,北京,2000年。
30.李永明,赵玉琪等主编。《实验分子生物学方法手册》。科学出版社,北京,1998年。
31.姜泊,张亚历等主编。《分子生物学常用实验方法》。人民军医出版社,北京,1996年。
32. Zeng M,Smith SK,Siegel F,et al.AdEasy system made easier by lecting the viral backbone plasmid preceding homologous recombination. Biotechniques.2001;31(2):260-62.
33.鄂征主编。《组织培养技术》。人民卫生出版社,北京,1986年。
34.司徒镇强,吴军正主编。《细胞培养》。世界图书出版公司,北京,1996年。
35. Kirshenbaum LA, MacLellan WR, Mazur W,et al. Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus. J. Clin Invest.1993 92: 381-387.
36. Perrella MA, Yet SF. Role of heme oxygenase-1 in cardiovascular function. Curr Pharm Des,2003,9:2479-248.
37. Naglaa KI, Andrew D, Gregory YH, et al. Hemoxygenase-1 in cardiovascular disease. J Am Coll Cardiol, 2008, 52: 971-978.
38. Cao J, Inoue K, Li X, Drummond G ,et al. Physiological significance of heme oxygenase in hypertension. Inter J biochemistry & cell biology, 2009,41:1025-33.
39. Joseph FN, Ashok JD. Upregulating the heme oxygenase system suppresses left ventricular hypertrophy in adult spontaneously hypertensive rats for 3 months.J Cardiac Fail, 2009,12:1-13.
40. Tang, YL et al. Protection from ischemic heart injury by a vigilant heme oxygenase-1 plasmid system. Hypertension, 2004, 43:746-751.
41. Muller, OJ et al. Improved cardiac gene transfer by transcriptional and transductional targeting ofadeno-associated viral vectors.Cardiovascular Research, 2006,70:70-78.
42. LI jianjun, LI Gengshan, Huang Congxin,et al.Comparative study of catheter-mediated gene transfer into heart.Chin Med J, 2002, 115:612-613.
43. Masamichi K, Dean M. Anselmo, RW,et al. A novel strategy against ischemia and reperfusion injury: cytoprotection with heme oxygenase system. Transplant Imm 2002,9:227-233
44. Barr E, Carroll J, Kalynych, A.M,et al. Efficient catheter-mediated gene transfer into the heart using replication-defective adenovirus. Gene Ther, 1994,1: 51-58.
45. Luo D. A new solution for improving gene delivery. Trends Biotechnol. 2004;22(3): 101-3.
46. Read ML, Logan A. 11th annual congress of the European Society of Gene Therapy. Exper Rev Antican Ther. 2004;4(1):49-51.
47. Grill J, Geoerger B, Lamfers M, et al. Conditionally replicative adenoviruses: a second wind for cancer gene therapy. Bull Cancer. 2003;90(12):1039-48.
48. Baum BJ, Goldsmith CM, Kok MR, et al. Advances in vector-mediated gene transfer. Immunol Lett. 2003;90(2-3):145-9.
49. Fukuhara H, Hayashi Y, Yamamoto N, et al. Improvement of transduction efficiency of recombinant adenovirus vector conjugated with cationic liposome for human oral squamous cell carcinoma cell lines.Oral Oncol. 2003 ;39(6):601-9.
50. Hedman M, Hartikainen J, Syvanne M,et al. Safety and feasibility of catheter-based local intracoronary vascular endothelial growth factor gene transfer in the prevention of postangioplasty and in-stent restenosis and in the treatment of chronic myocardial ischemia: phase II results of the Kuopio Angiogenesis Trial (KAT).Circulation. 2003 ;107(21):2677-83.
51. Hubberstey AV, Pavliv M, Parks RJ. Cancer therapy utilizing an adenoviral vector expressing only E1A.Cancer Gene Ther. 2002 ;9(4):321-9.
52. Danos O, Mullligan RC. Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc Natl Acad Sci USA. 1988;85:6460-64.
53. Mulligan RC. The basic science of gene therapy. Science. 1993;260:926-932.
54. Miller DG, Adam MA, Miller AD. Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol. 1990;10: 4239-42.
55. Friedman T, Yee JK. Pseudotyped retroviral vectors for studies of human gene therapy. Nature Med. 1995;1: 275-77.
56. Shashikant CS, Ruddle FH. Impact of transgenic technologies on functional genomics.Curr Issues Mol Biol. 2003;5(3):75-98.
57. Grimes BR, Warburton PE, Farr CJ. Chromosome engineering: prospects for gene therapy. Gene Ther. 2002;9(11):713-8.
58. Wong LH, Saffery R, Choo KH. Construction of neocentromere-based human minichromosomes for gene delivery and centromere studies.Gene Ther. 2002 ; 9(11):724-6.
59. Saffery R, Choo KH. Strategies for engineering human chromosomes with therapeutic potential.J Gene Med. 2002 ;4(1):5-13.
60. Park J,Ries J,Gelse J,et al.Bone regeneration in critical sized effects by cell mediated BMP2 gene transfer:acomparison of adenoviral vectors and liposomes.Gene Ther. 2003,10(13):1089 -98.
61. Ramesh R,Saeki T,Templeton NS,et al.Successful treatment of primary and disseminated human lung cancers by systemic delivery tumour suppressor genes using an improved liposome vector.Mol Ther. 2001,3(3):337-50.
62. MizuguchiH,KayMA.Efficient construction of a recombinant adenovirus vector by an improved in vitro ligation method.Hum Gene Ther.1998;9(17):2577-83.
63. Mark CA, Patel SR, Schwarz EA, et al. Biologic bypass with the use of adenovirus-mediated gene transfer of the complementary deoxyribonucleic acid for vascular endothelial growth factor 121 improves myocardial perfusion and function in the ischemic porcine heart. J Thorac Cardiovasc Surg,1998,115:168-177.
64. Brien ER, Alpers CE, Stewart DK, et al. Proliferation in primary and restenotic coronary atherectomy tissue:Implication for antiproliferative therapy. Circ Res,1993,73:223-231.
65. Pickering JG, Weir L, Jekanowsky J, et al. Proliferative activity in peripheral and coronary atherosclerotic plaque among patients undergoing percutaneous revascularization. J Clin Invest,1993,91:1469-1480.
66. Prentice H, Bishopric NH, Hick RN, et al. Regulated expression of a foreign gene targeted to ischemic myocardium. Cardiovasc Res, 1997,35: 567-574.
67. Miller DG, Adam MA, Miller AD,et al. Gene transfer by retrovirus vector occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol, 1990,10:4239-4242.
68. Brody Sl, Crystal RG, Boris-Lawrie K, et al. Adenovirus-mediated in vivo gene transfer. Ann NY Acad Sci,1997,716:90-103.
69. Flugelman MY, Jaklitsch MT, Newman KD, et al. Low level in vivo gene transfer into the arterial wall through a perforated balloon catheter. Circ, 1992,85:1110-1117.
70. Riessen R, Rahimizadeh H,T akeshita S, et al. Successful vascular gene transfer using a hydrogel coated balloon angioplasty catheter. Hum Gene Ther, 1993,4:749-758.
71. Lemarchand P, Jones M, Yamada I, et al. In vivo gene transfer and expression in normal uninjured blood vessels using replication-deficient recombinant adenovirus vectors. Circ Res, 1993,72:1132-1138.
72. Sommer B, Kuznetsov SA, Robey PG, et al. Efficient gene transfer into normal human skeletal cells using recombinant adenovirus and conjugated adenovirus-DNA complex. Calcif Tissue Int. 1999,64:45-51.
73. Wilson JM. Adenoviruses as gene delivery vehicles. N Engl J Med. 1996;334: 1185-1187.
74. Harary L, Farley B. In vitro studies of single isolated beating heart cells.Science, 1960, 131:16741675.
75. Simpson P, Savion S.Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cells.Cross-striations, ultrastructure,and chronotropic response to isoproterenol.Circ Res,1982,50:101-116.
76. Komuro I, Kaida T,Shibazaki Y,et al.Stretching cardiac myocytes stimulates protooncogene expression.J Biol Chem,1990,265:3595-3598.
77.司徒镇强,吴军正主编。《细胞培养》第一版。西安,世界图书出版公司,2004,250-252。
78.蔡文琴主编。《现代实用细胞与分子生物学实验技术》。人民军医出版社,北京,2003年。
79.颜子颖,王海林编译。《精编分子生物学指南》。科学出版社,北京,1998。
80.黄培堂,俞炜源,陈添弥等译。《PCR技术实验指南》。科学出版社,北京,1999年。
81. Ferrier GR,Moffat MP,Lukas A.Possible mechanisms of ventrieular arrhythmiaselieited by ischemia followed by reperfusion.Cire Res.1985:56:184.
82. Koyama T,Temma K,Akera T.Reperfusion-induced contracture develops with a decreasing [Ca2+]:in single heart cells.Am J physiol.1991,261:Hlll5.
83. Chen XW, Zhang XY, Kubo H, etal. Ca2+ infux- induced sarcoplasmic reticulum Ca2+ overload causes mitochondrial-dependent apoptosis inventricular myoeytes.Circ Res,2005,97(11):1009-1017.
84. Altavilla D,Dendato B,Campo GM,etal.IRFI042,a novel dual vitamin E-like antioxidant,inhibits activation of nuclear factor-KB and reduces the inflammatory response in myocardial ischemia-reperfusion injury.Cardiovasc Res, 2000, 47(3): 515-528,
85. Thandroyen FT, Bellotto D,Katayama A,et al.Subcellular electrolyte alterations during progressive hypoxia following reoxygenation in isolated neonatal rat ventricular myocytes.Cire Res,1992,71(l):106-112.
86. Kuramochi T,Honda M,Tanaka K,et al.Contrasting effects of an angiotensin converting enzyme inhibitor and a calcium antagonist on calcium transients in isolated rat cardiac myocytes.Cardiovasc Res,1994;28:1407-1413.
87. Folkman J.Tumor angiogenesis:therapeutic implications.N Engl J Med.1971;285: 1182–1186.
88. Okamoto N,Tobe T,Hackett SF,Ozaki H,et al.Animal model:transgenic mice with increased expression of vascular endothelial growth factor in the retina:a new model of intraretinal and subretinal neovascularization.Am J Pathol.1997;151:281-291.
89. Baffi J,Byrnes G,Chan CC, et al.Choroidal neovascularization in the rat induced by adenovirus mediated expression of vascular endothelial growth factor.Invest Ophthalmol Vis Sci.2000;41:3582-3589.
90. Frank RN,Amin RH,Eliott D,Puklin JE,Abrams GW.Basic fibroblast growth factor and vascular endothelial growth factor are present inepiretinal and choroidal neovascular membranes.Am J Ophthalmol.1996;122:393-403.
91. Hamm CW,Katus HA.New biochemical markers for myocardial cell injury.Curr Opin Cardiol,1995;10(4):355-360.
92. Hangaishi M,Ishizaka N,Aizawa T,et al.Induction of heme oxygenase-1 can act protectively against cardiac ischemia reperufusion in vivo.Bioehem Biophys ResCommun,2000:279:582-588.
93. Clark , JE,Foresti R,Sarathchandra P, et al.Heme oxygenase-1 derived bilirubin ameliorates postischemic myocardial dysfunetion.Am J Physio1 Heart Circ Physio1,2000;278:643-651.
94. Gottlieb RA,Burleson KO,Kloner RA,et al.Reperfusion injury induces apoptosis in rabbit cardiomyocytes.J Clin Invest,1994;94(4):1621-1628.
95. Henry F,Deborah G.Apoptosis in ischemic and reperfusion rat myocardium.Circ Res,1996;79(5):949-956.
96. Brown WM,Hotsley WS,Gott JP,et al.Myocardial protection for coronary artery in patients with acute myocardial infarction:resuscitation of ischemic myocardium.Semin in Thorac and Cardiovasc Surg,1995;7(4):191-197.
97. Sharber J.Oxygen radicals and calcium ion flux:role in reperfusion injury following AMI.Dimens Crit Care Nurs,1994;13(5):256-261.
98. Flitter WD.Free radicals and myocardial reperfusion injury.Br Med Bull,1993; 49(3):545-555.
99. Kirshenbaum LA,Singal PK.Changes in antioxide enzyme in isolated cardiac myocyte subjected to hypoxia-reoxygenation.Lab Invest,1992,67(6):796-803.
100. Xie Y,Zhu WZ,Zhu Y,et al.Intermittent high altitude hypoxia protects the heart against lethal Ca2+ overload injury.Life Science,2004; 76(5):559-572.
101. Petrosillog, Ruggierofm, Divenosan, et al.Decreased complex III activity in mitochondria isolated from rat heart subjected to ischemia and reperfusion: role of reactive oxygen species and cardiolipin . FASEB J,2003, 17 (6): 714-716.
102. Goldhaber JI, Qayyum MS. Oxygen free radicals and excitation-contraction coupling . Antioxid Redox Signal, 2000;2 (1): 55-64.
103. Xu B,Dong GH,Liu H, et al.Recombinant human erythropoietin pretreatment attenuates myocardial infarct size:a possible mechanism involves heat shock protein 70 and attenuation of nuclear factor-kappa B.Ann Clin Lab Sci,2005;35(2):161-168.
104. Chandrasekar B,Smith JB,Freeman GL.Ischemia-reperfusion of rat myocardium activates nuclear factor-KB and induces neutrophil infiltration via lipopolysaccharide-induced CXC chemokine.Circulation,2001;103(18):2296-2302.
105. Date T,Belanger AJ,Mochizuki S.Adenovirus-mediated expression of P35 preventshypoxia reoxygenation injury by redueing reactive oxygen species and caspase activity.Cardiovasc Res,2002;55(2):309-319.
106.方喜业,《医学实验动物学》。北京:人民卫生出版社,1995,291-295。
107. Wright MJ,Wightman LML,Latchman DS,et al.In vivo myocardial gene transfer:optimization and evaluation of intracoronaru gene delivery in vivo[J].Gene Therapy,2001;8:1833-1839.
108. FlissH,GattingerD.Apoptosis in ischemic and reperfused rat myocardium.Circ Res,1996;79(5):949-956.
109. Brounwald主编,陈灏珠主译,《心脏病学》第五版。人民卫生出版社,2001。
110.金惠铭主编,《病理生理学》第五版。人民卫生出版社,2000年,152-160。
111. Murry C,Jennings R,Reimer K. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation. 1986; 74: 1124-1136.
112. Li J-J,Ueno H,Pan Y,et al.Percutaneous transluminal gene transfer into canine myocardium in vivo by replication-defective adenovirus. Cardiovasc Res.1995;30: 97-105.
113. Ferry N,Heard J. Liver-directed gene transfer vectors. Human Gene Therapy.1998; 9: 1975-1978.
114. Nabel EG,Pompili VJ,Plautz GE,et al. Gene transfer and vascular disease. Cardiovasc Res.1994;28: 445-454.
115. Qin L,Chavin KD,Ding Y,et al.Multiple vectors effectively achieve gene transfer in a murine cardiac transplantation model.Immunosuppression with TGF-1 or vIL-10.Transplantation.1995;59(6):809-816.
116. Donehue JK, Kikkawa K,Tomas AD,et al. Acceleration of widespread adenoviral gene transfer to intact rabbit hearts by coronary perfusion with low calcium and serotonin. Gene Ther.1998;5:630-634.
117. Davidson MJ,Jones JM,Emani S,et al. Cardiac gene delivery with cardiopulmonary bypass.Circulation.2001;104:131-133.
118. Katori M,Buelow R,Ke B,et al.Heme oxygenase-1 overexpression protects rat hearts from cold ischemia reperfusion injury via an antiapoptotic pathway.Transplantation. 2002;73(2): 287-292.
119. Katori M, Busuttil RW, Kupiec-Weglinski J. Heme oxygenase-1 System in organtransplantation. Transplantation, 2002, 74: 905-912.
120. Alok SP, Luis G. Melo, et al. Chronic recurrent myocardial ischemic injury is significantly attenuated by pre-emptive adeno-associated virus heme oxygenase-1 gene delivery. J Am Coll Cardiol 2006;47:635- 43.
121. Yoshida T, Maulik N, Ho YS, Alam J, Das DK. H(mox-1) constitutes an adaptive response to effect antioxidant cardioprotection: a study with transgenic mice heterozygous for targeted disruption of the Heme oxygenase-1 gene. Circulation. 2001;103:1695–701.
122. Becker LB. New concepts in reactive oxygen species and cardiovascular reperfusion physiology. Cardiovasc Res.2004;61:461-70.
123. Sharma HS,Das DK,Verdouw PD. Enhanced expression and localization of heme oxygenase-1 during recovery phase of porcine stunned myocardium. Mol Cell Biochem.1999;196:133-139.
124. Lee TS,Chan LY. Heme oxygenase-1 mediates the anti-inflammatory effect of interleukin-10 in mice. Nat Med.2002;8:240-246.
125. Choi AM.Heme oxygenase-1 protects the heart[J].Circ Res.2001;89:168-173.
126. Maines MD,Panahian N.The heme oxygenase system and cellular defense mechanisms. Do HO-1 and HO-2 have different function? Adv Exp Med Biol. 2001;502:249-272.
127. Hori R,Kashiba M,Toma T,et al.Gene transfection of H25A mutant heme oxygenase-1 protects cells against hydroperoxide-induced cytotoxicity. J Biol Chem.2002;277: 10712-10718.
128. Frangogiannis NG,Smith CW,Entman ML.The inflammatory response in myocardial infarction.Cardiovasc Res.2002;53:31-47.
129. Sun B,Fan H,Honda T,et al.Activation of NF kappa B and expression of ICAM-1 in ischemic-reperfused canine myocardium.J Mol Cell Cardiol.2001;33:109-119.
130. Vachharajani TJ,Work J,Issekutz AC,et al.Heme oxygenase modulates selectin expression in different regional vascular beds.Am J Physiol Heart Circ Physiol.2000;278:H613-617.
131. Hayashi S,Takamiya R,Yamaguchi T,et al.Induction of heme oxygenase-1 suppresses venular leukocyte adhension elicited by oxidative stress:role of bilirubin generated bythe enzyme.Circ Res.1999;85:663-671.
132. Yet SY,Tian R,Layne MD,et al.Cardiac specific expression of heme oxygenase-1 protects against ischemia and reperfusion injury in transgenic mice.Circ. 2001;89:105-107.
133. Wang R,Wu LY.The chemical modification of K-Ca channels by carbon monoxide in vascular smooth muscle cell.J Biol Chem.1997;272:8222-8226.
134. Peng J,Lu R,Ye F,et al.The heme oxygenase-1 pathway is involved in calcitonin gene-related peptide-mediated delayed cardioprotection induced by monophosphoryl lipid A in rats.Regul Pept.2002;103:1-7.
135. Alok SP, Anthony S, Patricia MD et al. Heme-oxygenase-1-induced protection against hypoxia/reoxygenation is dependent on biliverdin reductase and its interaction with PI3K/Akt pathway. J Mol Cell Cardiol. 2007,43: 580-592.
136. Brouard S,Berberat PO,Tobiasch E,et al.Heme oxygenase-1 derived carbon monoxide requires the activation of transcription factor NF-kappa B to protect endothelial cell from tumor necrosis factor-alpha-mediated apoptosis.J Biol Chem.2002;277: 17950-17961.
137. Zweier JL,Fertmann J,Wei G.Nitric oxide and peroxynitrite in postichemic myocardium.Antioxid Redox Signal.2001;3:11-22.
138. Maines MD,Panahian N.The heme oxygenase system and cellular defense mechanisms. DO HO-1 and HO-2 have different function? Adv Exp Med Biol.2001;502:249-272.
139. Hartsfield CL.Cross talk between carbon monoxide and nitric oxide. Antioxid Redox Signal.2002;4:301-307.
140. Kamilla K, Kevin D. Approaches for cardiovascular gene therapy in animal models. Drug Discovery Today: Disease Models.2006,3:305-311.
1. Otterbein LE, Choi AMK. Heme oxygenase: colors of defense against cellular stress.Am J Physiol,2000,279:L1029-L1037.
2. Motterlini R, Foresti R, Intaglietta M, et al. NO-mediated activation of heme oxygenase: endogenous cytoprotection against oxidative stress to endothelium. Am J Physiol Heart Circ Physiol.1996;270: H107-114.
3. Durante W, Kroll MH, Christodoulides N, et al. Nitric oxide induces heme oxygenase-1 gene expression and carbon monoxide production in vascular smooth muscle cells. Circ Res. 1997; 80:557-564.
4. Tian W, Bonkovasky HL, Shibahara S, et al. Urea and hypertonicity increase expression of heme oxygenase-1 in murine renal medullary cells. Am J Physiol Renal Physiol. 2001; 281: F983-991.
5. Chen YH, Lin SJ, Lin MW, et al. Microsatellite polymorphism in promoter of heme oxygenase-1 gene is associated with susceptibility to coronary artery disease in type 2 diabetic patients. Hum Genet. 2002; 111:1-8.
6. Schillinger M, Exner M, Mlekusch W, et al. Heme oxygenase-1 genotype is a vascular anti-inflammatory factor following balloon angioplasty. J Endovasc Ther. 2002; 9:385-394.
7. Otterbein LE, Bach FH, Alam J, et al. Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat Med. 2000; 6: 422-428.
8. Liu XM., Chapman GB., Peyton KJ, et al. Carbon monoxide inhibits apoptosis in vascular smooth muscle cells. Cardiovasc. Res.2002; 55: 396–405
9. Liu XM, Peyton KJ, Ensenat D, et al. Endoplasmic Reticulum Stress Stimulates Heme Oxygenase-1 Gene Expression in Vascular Smooth Muscle. J Biol Chem. 2005; 280:872-877.
10. McDaid J, Yamashita K, Chora A, et al. Heme oxygenase-1 modulates the allo-immune response by promoting activation-induced cell death of T cells. FASEB J. 2005; 19:458-460.
11. Dasilva JL, Tiefenthaler M, Park E, et al. Tin-mediated heme oxygenase gene activationand cytochrome P450 arachidonate hydroxylase inhibition in spontaneously hypertensive rats. Am J Med Sci.1994;307:173 181.
12. Christou H, Morita T, Hsieh CM, et al. Prevention of hypoxia-induced pulmonary hypertension by enhancement of endogenous heme oxygenase-1 in the rat. Circ Res. 2001; 86:1224–1229.
13. Johnson RA, Lavesa M, Deseyn K, et al. Heme oxygenase substrates acutely lower blood pressure in hypertensive rats. Am Phys Soci. 1996; 271:H132-138.
14. Foresti R, Goatly H, Green CJ, et al. Role of heme oxygenase-1 in hypoxia reoxygenation:requirement of substrate heme to promote cardioprotection.Am J Physiol Heart Circ Physiol.2001;281(5):H1976-984.
15. Wu TW, Wu J, Li RK, et al. Albumin-bound bilirubins protect human ventricular myocytes against oxyradical damage. Biochem Cell Biol. 1991; 69: 683– 688.
16. Balla G, Jacob HS, Balla J, et al.Ferritin:a cytoprotective antioxidant strategem of endothelium. J Biol Chem,1992;267:18148-18153.
17. Kaur H,Hughes MN, Green CJ, et al. Interaction of bilirubin and biliverdin with reactive nitrogen species. FEBS Lett, 2003;543 (1-3):113-119.
18. Mancuso C,Bonsignore A, Stasio ED, et al. Bilirubin and S-nitrosothiols interaction:evidence for a possible role of bilirubin as a scavenger of nitric oxide.Biochem Pharmacol,2003;66 (12):2355-2363.
19. Hayashi S, Takamiya R, Yamaguchi T, et al. Induction of heme oxygenase-1 suppresses venular leukocyte adhesion elicited oxidative stress:role ofbilirubin generated by the enzyme. Circ Res,1999;85:663-671.
20. Wagener F,Silva JL, Farley T, et al. Differential effects of heme oxygenase isoforms on heme mediation of endothelial intracellular adhesion molecule 1 expression. Pharmacol Exp Ther,1999;291:416-423.
21. Otterbein LE, Bach FH, Alam J,et al. Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway.Nat Med, 2000;6 (4):422-428.
22. Katori M, Buelow R, Ke B, et al.Heme oxygenase-1 overexpression protects rat hearts from cold ischemia reperfusion injury via an antiapoptotic pathway.Transplantation, 2002;73 (2): 287-292.
23. Akamatsu Y, Haga M, et al. Heme oxygenase-1derived carbonmonoxide protectshearts from transplant-associated ischemia reperfusion injury.FASEB J, 2004;10. 1096-1099.
24. Vulapalli SR, Chen ZY, Chua BHL,et al. Cardioselective overexpression of HO-l prevents I/R-induced cardiac dysfunction and apoptosis.Am J Physiol Heart Circ Physiol.2002;283 (2):H688-694.
25. Melo LG, Agrawal R, Zhang L, et al. Gene therapy strategy for long-term myocardial protection using adeno-associated virus-mediated delivery of heme oxygenase gene. Circulation. 2002; 105:602–607.
26. Foresti R, Sarathchandra P, Clark JE, et al. Peroxynitrite induces heme oxygenase-1 in vascular endothelial cells: a link to apoptosis. Biochem J, 1999;339(Pt 3):729-736.
1. French BA, Mazur W, Geske RS, et al. Direct in vivo gene transfer into porcine myocardium using replication-deficient adenoviral vectors[J]. Circ, 1994,90:2414-2424.
2. Berkner KL. Expression of heterologous sequences in adenoviral vectors[J]. Curr Top Microbiol Immunol,1992,158:39-66.
3. Fallaux FJ, Kranenburg O, Cramer SJ, et al. Characterization of 911:A new helper cell line for the titration and propagation of early-region-deleted adenoviral vectors[J]. Hum Gene Ther, 1996,7:215-222.
4. Mark CA, Patel SR, Schwarz EA, et al. Biologic bypass with the use of adenovirus-mediated gene transfer of the complementary deoxyribonucleic acid for vascular endothelial growth factor 121 improves myocardial perfusion and function in the ischemic porcine heart[J]. J Thorac Cardiovasc Surg, 1998,115:168-177.
5. O‘Brien ER, Alpers CE, Stewart DK, et al. Proliferation in primary and restenotic coronary atherectomy tissue:Implication for antiproliferative therapy[J]. Circ Res,1993,73:223-231.
6. Pickering JG, Weir L, Jekanowsky J, et al. Proliferative activity in peripheral and coronary atherosclerotic plaque among patients undergoing percutaneous revascularization[J]. J Clin Invest,1993,91:1469-1480.
7. Prentice H, Bishopric NH, Hick RN, et al. Regulated expression of a foreign gene targeted to ischemic myocardium[J]. Cardiovasc Res, 1997,35: 567-574.
8. Miller DG, Adam MA, Miller AD,et al. Gene transfer by retrovirus vector occurs only in cells that are actively replicating at the time of infection[J]. Mol Cell Biol, 1990,10:4239-4242.
9. Brody Sl, Crystal RG, Boris-Lawrie K, et al. Adenovirus-mediated in vivo gene transfer[J]. Ann NY Acad Sci,1997,716:90-103.
10. Flugelman MY, Jaklitsch MT, Newman KD, et al. Low level in vivo gene transfer into the arterial wall through a perforated balloon catheter[J]. Circ, 1992,85:1110-1117.
11. Riessen R, Rahimizadeh H,T akeshita S, et al. Successful vascular gene transfer using a hydrogel coated balloon angioplasty catheter[J]. Hum Gene Ther, 1993,4:749-758.
12. Lemarchand P, Jones M, Yamada I, et al. In vivo gene transfer and expression in normal uninjured blood vessels using replication-deficient recombinant adenovirus vectors[J]. Circ Res, 1993,72:1132-1138.
13. Sommer B, Kuznetsov SA, Robey PG, et al. Efficient gene transfer into normal humanskeletal cells using recombinant adenovirus and conjugated adenovirus-DNA complex[J]. Calcif Tissue Int. 1999,64:45-51.
14. Smith TA, Mehaffey MG, Kayda DB, et al. Adenovirus mediated expression of therapeutic plasma levels of human factorⅨin mice[J]. Nature Genet, 1993,5:397-402.
15. Lee LY, Patel SR, Hackett NR, et al. Focal angiogen therapy using intramyocardial delivery of adenovirus vector coding for vascular endothelial growth factor 121[J]. Ann Thorac Surg,2000,69:14-24.
16. Wang Q, Finer MH. Second-generation adenovirus vectors[J]. Nature Med, 1996,2:714-716.
17. Shean MK, Baskin G, Sullivan D, et al. Immunomodulation and adenoviral gene transfer to lungs of nonhuman primate[J]. Hum Gen Ther,2000,11:1047-1055.
18. Newman KD, DunnPF, Owens JL, et al. Adenovirus-mediated gene transfer into normal rabbit arteries results in prolonged vascular cell activation, inflammation and neointimal hyperplasia[J]. J Clin Invest, 1995,95:2955-2965.
19. Gao GP,Yang Y,Wilson JM.Biology of adenovirus vectors with E1 and E4 deletions for liver directed gene therapy[J]. J Virol,1996,70:8934-8943.
20. Amalfitano A,Hauser MA,Hu H,et al Production and characterization of improved adenovirus vectrors with the E1,E2 and E3 genes deleted[J]. J Virol,1998,72:926-933.
21. Lusky M, Christ M, Rittner K,et al. In vitro and in vivo biology of recombinant adenovirus vectors with E1,E1PE2A,or E1PE4 deleted[J]. J Virol,1998,72:2022-2032.
22. Hu H, Serra D, Amalfitano A. Persistence of an [ E12,polymerase2] adenovirus vector despite transduction of a neoantigen into immune competent mice[J]. Hum Gene Ther,1999, 10:355-364.
23. Morsy MA, Gu MC,Motzel S,et al. An adenoviral vector deleted for all viral coding sequences results an enhancde safety and extended expression of a leptin transgene[J]. Proc Natl Acad Sci USA,1998,95:7866-7871.
24. Kochanek S, Clemens PR, Mitani K, et al. A new adenoviral vector:replacement of all viral coding sequences with 28 kb of DNA independently expressing both full2length dystrophin and beta-galactosidase[J]. Proc Natl Acad Sci USA,1996,93:5731-5736.
25. Lieber A,He CY,Kirillova I,et al. Recombinant adenoviruses with large deletions generated by cre2 mediated excision exhibit different biological properties compared with first generation vectors in vitro and in vivo[J]. J Virol,1996,70:8944-8960.
26. Svensson EC,Marshal DJ,Woodard K,et al. Efficient and stable transduction of cardiomyocytes after intramyocardial injection or intracoronary perfusion with recombinantadeno-associated virus vectors[J]. Circulation, 1999,99: 201-205.
27. Rolling F,Nong Z,Pisvin S,et al.Adeno-associated virus-mediated gene transfer into rat carotid arteries[J]. Gene Ther, 1997,4: 757-761.
28. 281.Felgner PL, Barenholz Y, Behr JP,et al.Nomenclature for synthetic gene delivery systems[J]. Hum.Gene Ther, 1997,8: 511-512.
29. Schwartz LB,Moawad J. Gene therapy for vascular disease[J].Ann Vasc Surg,1997,11:189- 199.
30. Kirshenbaum LA, MacLellan WR, Mazur W,et al. Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus[J].Clin. Invest, 1993,92: 381-387.
31. Kaplan JM, St George JA, Pennington SE, et al. Humoral and cellular immune responses to Ad2/CFTR-2[J].Gene Ther,1996,3( 2):117- 127.
32. Christ M, Lusky M, Stieckel F, et al. Gene therapy with recomhinant adenovirus vectors: evaluation of the host immune response[J].Immunol Lett,1997,57( 1997):19-25.
33. Yap T, Brien TO, Tazelaar HD, Immunosuppression prolongs adenoviral mediated transgene expression in cardiac allograft transplantation[J].Cardiovasc Res,1997,35: 529-535.
34. Dipak K, Richard M, Engelman, et al. Molecular targets of gene therapy[J].Ann Thor Surg,1999, 68(5):1929-1933.
35.杨惠玲等,《高级病理生理学》第一版。北京:科学出版社,1998,10:569 - 594.
36. Donahue JK, Kiklawa K,Johivs D,et al.Ultrarapid,highly efficient viral gene transfer to the heart[J]. Proc Natl Acad Sci, 1997, 94:4664-4668.
37. Donahue JK, Kiklawa K, Johivs D,et al.Acceleration of widespread adenoviral gene transfer to intact rabbit hearts by coronary perfusion with low calcium and serotonin[J]. Gene Therapy, 1998, 95:5251-5256.
38. Damien L, Hatem SN, Heimburger M,et al.How to optimize in vivo gene transfer to cardiac myocytes:mechanical or pharmacological procedures [J]? Human Gene Therapy, 2001, 12:1601-1610.
39. Wright MJ, Wightman LML, Latchman DS,et al.In vivo myocardial gene transfer:optimization and evaluation of intracoronaru gene delivery in vivo[J].Gene Therapy, 2001, 8:1833-1839.
40. Davidson MJ, Jones JM, Emani S,et al.Cardiac gene delivery with cardiopulmonary bypass [J]. Circulation, 2001,104:131-133.
41. Jones JM, Wilson KH, Kock WJ,et al.Adenovital gene transfer to the heart duringcardiopulmonary bypass:effect of myocardial protection technique on transgene expression[J]. Euro J Card-Thor Surg, 2002, 21:847-852.
42. LI jianjun, LI Gengshan, Huang Congxin,et al.Comparative study of catheter-mediated gene transfer into heart[J].Chin Med, J 2002, 115:612-613.
43. Riessen R. and Isner J.M. Prospects for site-specific delivery of pharmacologic and molecular therapies[J].Am. CollCardiol, 1994,23: 1234-1244.
44. Barr E, Carroll J, Kalynych, A.M,et al. Efficient catheter-mediated gene transfer into the heart using replication-defective adenovirus[J]. Gene Ther, 1994,1: 51-58.
45. Tomiyasu K, Oda Y, Nomura M,et al. Direct intra-cardiomuscular transfer of beta2-adrenergic receptor gene augments cardiac output in cardiomyopathic hamsters[J].Gene Ther, 2000,7: 2087-2093.
46. Lamping KG, Rios CD, Chun JA, et al. Intrapericardial administration of adenovirus for gene transfer[J].Am J Physiol, 1997,272: H310-H317 .
47. Neyroud N, Nuss HB, Leppo MK,et al.Gene delivery to cardiac muscle[J].Methods Enzymol., 2002,346: 323-334.
48. Feldman LJ. and Steg G. Optimal techniques for arterial gene transfer[J].Cardiovasc. Res., 1997,35: 391-404.
49. Valen G, Paulsson G, Vage J. Induction of inflammatory mediators during reperfusion of the human heart[J]. Ann Thorac Surg, 2001, 71: 22E232
50. Morishita R, Sugimoto T, Aoki M, et al. In vivo transfection of cis element "decoy" against nuclear factor-kappaB binding site prevents myocardial infarction[J]. Nat. Med., 1997,3: 894-899.
51. Sawa Y, Morishita R, Suzuki K ,et al.novel strategy for myocardialprotection using in vivo transfection of cis element 'decoy' against NF kappaB binding site: evidence for a role of NF kappaB in ischemia reperfusion injury[J]. Circulation, 1997,96: 2-4.
52. Kupatt C, Wichels R, Deiss M,et al. Retroinfusion of NF kappaB decoy oligonucleotide extends cardioprotection achieved by CD18 inhibition in a preclinical study of myocardial ischemia and retroinfusion in pigs[J]. Gene Ther, 2002,9: 518-526.
53. Matsui Y, Inobe M, Okamoto H,et al. Blockade of T cell costimulatory signals using adenovirus vectors prevents both the induction and the progression of experimental autoimmune myocarditis[J]. J. Mol. Cell Cardiol, 2002,34: 279-295.
54. Nakano A, Matsumori A, Kawamoto S, et al. Cytokine gene therapy for myocarditis by in vivo electroporation[J]. Hum. Gene Ther, 2001,12: 1289-1297.
55. Cull VS, Bartlett EJ and James CM. Type I interferon gene therapy protects against cytomegalovirus-induced myocarditis[J]. Immunology, 2002,106: 428-437.
56. Watanabe K, Nakazawa M, Fuse K, et al. Protection against autoimmune myocarditis by gene transfer of interleukin-10 by electroporation[J]. Circulation, 2001,104: 1098-1100
57. Lim BK, Choe SC, Shin JO, et al. Local expression of interleukin-1 receptor antagonist by plasmid DNA improves mortality and decreases myocardial inflammation in experimental coxsackieviral myocarditis[J].Circulation,2002,105: 1278-1281.
58. Bilbao G, Contreras JL, Gomez-Navarro J, et al. Genetic modification of liver grafts with an adenoviral vector encoding the Bcl-2 gene improves organ preservation[J]. Transplantation, 1999,67: 775-783.
59. Chen Z, Chua CC, Ho YS, et al. Overexpression of Bcl-2 attenuates apoptosis and protects against myocardial I/R injury in transgenic mice[J]. Am. J. Physiol. Heart Circ.Physiol, 2001,280: H2313-H2320
60. Kirshenbaum LA and de Moissac D. The bcl-2 gene product prevents programmed cell death of ventricular myocytes[J]. Circulation, 1997,96: 1580-1585.
61. Huang J, Ito Y, Morikawa M, Uchida H, et al. Bcl-xL gene transfer protects the heart against ischemia reperfusion injury[J]. Biochem Biophys Res Commun, 2003,311: 64-70.
62. Sawitzki B, Amersi F, Ritter T, et al. Upregulation of Bag-1 by ex vivo gene transfer protects rat livers from ischemia/reperfusion injury[J]. Hum Gene Ther, 2002,13: 1495-1504.
63. Laugwitz KL, Moretti A, Weig HJ, et al. Blocking caspase-activated apoptosis improves contractility in failing myocardium[J]. Hum. Gene Ther, 2001,12: 2051-2063.
64. Matsui T, Li L, del M, et al. Adenoviral gene transfer of activated phosphatidylinositol 3'-kinase and Akt inhibits apoptosis of hypoxic cardiomyocytes in vitro[J]. Circulation, 1999,100: 2373-2379.
65. Miao W, Luo Z, Kitsis RN, et al. Intracoronary, adenovirus-mediated Akt gene transfer in heart limits infarct size following ischemia-reperfusion injury in vivo[J]. J. Mol. Cell Cardiol, 2000,32: 2397-2402.
66. Lehmann TG., Wheeler M.D, Schoonhoven R, et al. Delivery of Cu/Zn-superoxide dismutase genes with a viral vector minimizes liver injury and improves survival after liver transplantation in the rat[J]. Transplantation, 2000,69: 1051- 1057
67. Abunasra HJ, Smolenski RT, Yap J, et al. Multigene adenoviral therapy for the attenuation of ischemia-reperfusion injury after preservation for cardiac transplantation[J]. J Thorac Cardiovasc Surg, 2003,125: 998-1006.
68. Wink DA, Hanbauer I, Krishna MC. Nitric oxide protects against cellular damage and cytotoxicity from reactive oxygen species[J].Proc Natl Acad Sci USA,1993,90:9813～9817.
69. Johnson G, Tsao PS, Mulloy D, et al. Cardioprotective effects of acidified sodium nitrite in myocardial ischemia with reperfusion[J].J Pharmacol Exp Ther,1990,252(1):35～41。
70. Suda T, Mora BN, D'Ovidio F, et al.In vivo adenovirus-mediated endothelial nitric oxide synthase gene transfer ameliorates lung allograft ischemia-reperfusion injury[J]. J Thorac Cardiovasc Surg, 2000, 119: 297-304.
71. Kim KS, Takeda K, Sethi R, et al. Protection from reoxygenation injury by inhibition of rac1[J]. J. Clin. Invest, 1998 ,101: 1821-1826.
72. Melo LG, Agrawal R, Zhang L,et al. Gene therapy strategy for long-term myocardial protection using adeno-associated virus-mediated delivery of heme oxygenase gene[J]. Circulation, 2002,105: 602-607.
73. Karp G. Cell and molecular biology: concepts and experiments. 2nd [M]. New York:John Wiley & ons Inc,1999, 204.
74. Kelly, S. et al. Gene transfer of HSP72 protects cornu ammonis 1 region of the hippocampus neurons from global ischemia: influence of Bcl-2[J]. Ann. Neurol. 2002,52:160–167.
75. Creagh EM, Carmody RJ, Cotter TG. Heat shock protein 70 inhibits caspase-dependent and -independent apoptosis in Jurkat T cells[J]. Exp Cell Res. 2000,257:58–66.
76. Wong HR, Ryan M Wispe JR. Stress response decreases NF-kappaB nuclear translation and increases 1-appaB alpha expression in A549 cells[J]. J Clin lnvest,1997,99(10):2423-2428
77. Ken Suzuki et al,.in vivo gene transfection with heat shock protein 70 enhances myocardial tolerance to ischemia-reperfusion in jury in rat[J]. J clin invest.1996,99(7),1645-1650
78. Jay jayakumar et al, Transfection of dornor hearts with heat shock protein 70 gene protects cardiac function against ischemia-reperfusion injury[J].Circulation.2000,88: 1264–1272.
79. Jayakumar J, Suzuki K, Sammut IA, et al. Heat shock protein 70 gene transfection protects mitochondrial and ventricular function against ischemia-reperfusion injury[J]. Circulation, 2001,104: 1303-1307.
80. Gripes CL, Watkins MW, Helmer G, et al. Angiogenic gene therapy (AGENT) trial in patients with stable angina pectoris[J]. Circulation,2002 .105:1291-1297.