| 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.
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