大鼠血红素加氧酶-1基因转染对大鼠心肌缺血再灌注损伤的影响

Effects of transfection of rat heme oxygenase-1 on myocardial ischemia-reperfusion injury in rats

目的评价重组腺相关病毒（rAAV）介导大鼠血红素加氧酶-1（rHO-1）基因转染在大鼠心肌缺血再灌注损伤的影响及其机制。方法健康雄性SD大鼠81只，体重220～280g，随机分为4组：假手术组（SH组，n=9）、生理盐水组（NS组，n=24）、重组腺相关病毒-荧光蛋白组（rAAV—EGFP组，n=24）和重组腺相关病毒-HO-1组（rAAV-HO-1组，n=24）。NS组、rAAV-EGFP组和rAAV-HO-1组分别心肌注射600μl生理盐水、rAAV-EGFP（1．5×10^11v．g．）或rAAV-HO-1（1．5×10^11v．g．）。在基因转染后3个月，NS组、rAAV-EGFP组和rAAV-HO-1组各处死3只大鼠，取注射部位心肌，通过测定心肌细胞荧光蛋白的表达，计算转染率，并通过免疫组化染色和RT-PCR法检测HO-1蛋白和HO-1 mRNA的表达。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；再灌注120min后，处死大鼠，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。结果心肌细胞转染率为（53．5±2．0）％。与NS组比较，rAAV-HO-1组HO-1蛋白及HO-1 mRNA表达增加（P〈0．01）；与rAAV-EGFP组比较，rAAV-HO-1组HO-1蛋白及HO-1 mRNA表达增加（P〈0．01）；与SH组比较，NS组和rAAV-EGFP组SOD活性下降，MDA含量升高（P〈0．01）；与NS组和rAAV-EGFP组比较，rAAV-HO-1组SOD活性升高，MDA含量降低（P〈0.01）。NS组及rAAV-EGFP组心肌细胞超微结构损伤较SH组和rAAV-HO-1组重。结论腺相关病毒介导的血红素加氧酶-1基因转染大鼠心肌细胞后，可减轻心肌缺血再灌注损伤，其机制与抗氧化有关。

Objective To determine the myocardial expression of rat heme oxygenase-1 （rHO-1） gene carried by recombinant adeno-associated virus （rAAV） in transfected hearts and to investigate the effect of rHO-1 on myocardial ischemia-reperfusion injury （I/R） and the underlying mechanism. Methods Eighty-one healthy male SD rats weighing 220-280 g were randomly divided into 4 groups ： group Ⅰ sham operation （ SH n = 9 ） ; group Ⅱ normal saline （NS n = 24） ; group Ⅲ rAAV-EGFP （ n = 24） and group Ⅳ rAAV-rHO-1 （ n = 24） . NS, rAAV-EGFP and rAAV-rHO-1 were transferred into normal rat hearts by intramyocardial injection in group Ⅱ , Ⅲ , and Ⅳ . Nine animals in each group were sacrificed 3 months after the gene delivery in group Ⅱ , Ⅲ , and Ⅳ . The expression of rHO-1 in the injected myocardium was determined by immuno-histochemistry and RT-PCR. EGFP-expression cardiomyocytes were counted on heart sections using direct fluorescence microscopy. The rest animals in the 4 groups were anesthetized, tracheostomized and mechanically ventilated. The left coronary anterior descending artery was occluded for 30 rain followed by 120 min reperfusion. The animals were killed at the end of 120 min reperfusion and their hearts were harvested for determination of myocardial SOD activity and MDA content and electron-microscopic examination of myocardium. Results The intramyocardial gene delivery led to （53.5 ± 2.0） % transduction. The expression of rHO-1 mRNA and protein was significantly higher in rAAV-rHO-1 group （group Ⅲ ） than in NS （ Ⅱ ） and rAAV-EGFP （ Ⅲ ） . The damage to myocardial ultrastructure was significant severe, SOD activity was significantly decreased and MDA content increased in NS and rAAV-EGFP groups than in SH group. The ultrastructure was well preserved, SOD activity was significantly higher and MDA content lower in rAAV-rHO-1 group （group Ⅳ ） than in group Ⅱ and Ⅲ. Conclusion The HO-1 gene carried by AAV has protective effect on myocardium against I/R injury. Its antioxidant properties may be involved in the mechanism,