大鼠血红素加氧酶-1基因转染对心肌缺血再灌注损伤大鼠炎性因子的影响

Effects of transfection of rat heme oxygenase-1 gene on inflammatory mediators induced by myocardial ischemia/reperfusion injury in rats

目的探讨重组腺相关病毒（rAAV）介导大鼠血红素加氧酶-1（HO-1）基因转染对心肌缺血再灌注损伤大鼠炎性因子的影响。方法雄性SD大鼠93只，体重225．275g，随机分为4组：假手术组（SH组，n=12）、生理盐水组（NS组，n=27）、重组腺相关病毒．荧光蛋白组（rAAv-EGFP组。n=27）及重组腺相关病毒．血红素加氧酶-1组（rAAV-rHO-1组，n=27）。NS组、rAAV-EGFP组和rAAV-rHO-1组分别于左心室前后壁共取4点注入600μl生理盐水、rAAV-EGFP或rAAV-HO-1病毒液。在基因转染后3个月，通过RT-PCR和Westernblot法检测HO-1mRNA和蛋白的表达，荧光显微镜下观察荧光蛋白的表达。采用结扎左冠状动脉前降支30min、再灌注120min的方法建立大鼠心肌缺血再灌注模型，再灌注120min后，处死大鼠，测定心肌梗死面积及血清TNF-α、IL-6水平，电镜下观察心肌细胞的超微结构。结果rAAV-rHO-1组HO-1表达明显高于NS组和rAAV-EGFP组（P〈0．01），仅rAAV-EGFP组心肌观察到荧光蛋白的表达；与SH组比较，NS组、rAAV-EGFP组和rAAV-rHO-1组心肌梗死面积增加，血清TNF-α、IL-6水平明显升高（P〈0．05），心肌结构紊乱；与NS组和rAAV-EGFP组比较，rAAV-rHO-1组心肌梗死面积减少（P〈0．01），血清TNF-α、IL-6水平明显降低（P〈0．05），心肌结构破坏程度较轻。结论重组腺相关病毒介导大鼠血红素加氧酶-1基因转染大鼠心肌细胞后，降低炎性因子的产生，从而减轻心肌缺血再灌注损伤。

Objective To investigate the effect of rat heme oxygenase-1 （rHO-1） gene carried by recombinant adeno-associated virus （rAAV） on myocardial ischemia/reperfusion injury （I/R） in rats. Methods Ninety-three male SD rats weighing 225-275 g were randomly divided into 4 groups ： group Ⅰ sham operation （ n = 12） ; group Ⅱ normal saline （NS） ＋ I/R （ n = 27） ; group Ⅲ rAAV-EGFP ＋ I/R （ n = 27） and group Ⅳ rAAV- rHO-1 ＋ I/R （n = 27）. NS, rAAV-EGFP and rAAV-rHO-1 600/11 were injected intra-myocardially at 4 sites along the left anterior descending branch of coronary artery （LAD） on the anterior and posterior walls of left ventricle in groupⅡ , Ⅲ and Ⅳ respectively. I/R was induced by occlusion of LAD for 30 min followed by 120 min reperfusion. At the end of 120 min reperfusion the hearts were removed for determination of myocardial infarct size and ultrastruemre examination. The serum concentrations of TNF-α and IL-6 were also measured. Results The HO-1 expression in rAAV-rHO-1 group （Ⅳ） was significantly higher than that in NS （ Ⅱ ） and rAAV-EGFP group （ Ⅲ ）. The expression of GFP was detected in rAAV-EGFP group only. The infarct size was significantly larger, the serum TNF-α and IL-6 concentrationss were significantly higher and the myocardial ultrastructure was significantly severer damaged in NS （ Ⅱ ）, rAAV-EGFP （ Ⅲ ） and rAAV-rHO-1 （ Ⅳ ） groups than in sham operation group （ Ⅰ ）. The infarct size was significantly smaller, the serum TNF-α and IL-6 concentrations were significantly lower and the myocardial ultrastructure was better-preserved in rAAV-rHO-1 group than in NS and rAAV-EGFP groups and there was no significant difference between NS and rAAH-EGFP groups. Conclusion rAAV-mediated rHO-1 gene transfection may attenuate myocardial I/R injury by inhibiting the production of inflammatory mediators in rats.