rAAV1-SERCA2a转导对慢性心力衰竭beagle犬的治疗作用

Therapeutic effects of adeno-associated virus gene mediated transfer of SERCA2a on beagle dogs with heart failure

目的研究重组1型腺相关病毒(rAAV1)介导的肌质网钙离子ATP酶2a(SERCA2a)基因转导对慢性心力衰竭(HF)beagle犬的治疗作用及可能的机制。方法经快速右心室起搏建立beagle犬的慢性HF模型。16只beagle犬分为4组:对照组(n=4),HF组(n=4),HF+EGFP组(n=4)和HF+SERCA2a组(n=4)。应用开胸心肌内注射术,分别将携带EGFP基因rAAV1和携带SERCA2a基因rAAV1导入HF+EGFP组和HF+SERCA2a组的beagle犬心肌,剂量为1×1012/ml,而对照组和HF组未进行任何处理。基因转导后30d,应用超声心动图、血流动力学检测各组beagle犬的心脏收缩和舒张功能、Western blotting检测SERCA2a蛋白表达水平,TUNEL法检测心肌细胞的凋亡指数,免疫组织化学染色检测Bax的表达。结果在基因转导后30d,HF+SERCA2a组SERCA2a蛋白表达水平较HF组明显增加,随着SERCA2a表达增加心功能也得到改善,左室收缩压峰值(LVSP)、左室舒张末压(LVEDP)、左室内压最大上升速率(LV+dp/dtmax)和左室内压最大下降速率(LV-dp/dtmax)均接近对照组水平,较HF组明显改善(P<0.05)。SERCA2a基因治疗后心肌细胞凋亡指数、Bax表达比HF组显著降低(P<0.05)。结论SERCA2a基因转导能够治疗慢性心力衰竭,改善心功能,抑制心肌细胞凋亡。

Objective To investigate the therapeutic effects of recombinant adeno-associated virual gene serotype 1（rAAV1）mediated transfer of sarcoplasmic reticulum Ca^2＋ ATPase（SERCA2a）on beagle dogs with heart failure（HF）.Methods The chronic HF model was reproduced in beagle dogs by giving rapid right ventricular pacing（230 beats/min）for 30 days.A reduced rate（180 beats/min）was continued for another 30 days.Sixteen beagle dogs were divided into four groups（4 each）;control group,HF group,HF＋EGFP group and HF＋SERCA2a group.After rapid pacing for 30 days,rAAV1-EGFP（1×10^12vg/ml） and rAAV-1SERCA2a（1×10^12vg/ml）were respectively delivered via intramyocardial routes,while no treatment was given to the animals in both control and HF groups.At the end of the study,haemodynamics,echocardiography and the protein expression of SRCA2a were measured respectively.The apotosis index of cardiac myocyte was evaluated by TUNEL.Bax expression was assessed by immunohistochemistry.Results After gene transfer of SERCA2a in HF beagle dogs for 30 days.the heart function was improved along with an increase in SERCA2a expression.Left ventricular systolic function was significantly increased,including the left ventricular systolic pressure（LVSP）,left ventricular maximal rate of pressure rise（LV＋dp/dtmax）,left ventricular maximal rate of pressure declin（LV-dp/dtmax,P〈0.05）,and LVEDP was significantly decreased （P〈0.05）.The apotosis indexes of cardiac myocyte,the protein expressions of Bax in HF group and HF＋EGFP group were significantly hgher than that in control group（P〈0.05）.Moreover,the apotosis index and Bax expression were significantly lower in HF＋SERCA2a group than that in HF group（P〈0.05）.Conclusion Gene transfer of SERCA2a can improve the cardiac function and inhibit the apoptosis of cardiac myocyte in beagle dogs with chronic heart failure.