同一滴度不同方法转染AAV9-SERCA2a基因对大鼠的有效性和毒副作用评价

Evaluation of the efficiency and toxicity of AAV9-SERCA2a gene transfection by different cardiac gene delivery methods at the same titer in rats

目的研究同一滴度不同心脏基因转染方法转染携带肌浆网钙ATP酶2a(sarcoplasmic reticulumCa2+ATPase 2a,SERCA2a)基因的9型腺相关病毒(adeno-associated virus serotype 9,AAV9)即AAV9-SERCA2a对SD大鼠心肌的有效性及对机体毒副作用的影响。方法体外成功构建AAV9-SERCA2a-EGFP病毒载体系统,90只雄性SD大鼠随机分为尾静脉注射(tail vein injection,TVI)组、心肌注射(intramyocardial injection,IMI)组、心包腔注射(intrapericardial injection,IPI)组,采用相应方法在体分别转染滴度1×1011vg/mL AAV9-SERCA2a-EGFP、AAV9-EGFP、NS各200μL。转染后30 d,荧光显微镜下观察心肝肾组织绿色荧光表达情况,Western blot检测SER-CA2a基因在大鼠各组织的相对表达量,体表12导联心电图检测心律失常发生率,HE染色观察病理学变化,心脏彩色多普勒超声评价心功能,血液生化学指标检测肝肾功能。结果三组左心室可见大量绿色荧光表达,IMI组荧光局限在注射点,三组肝肾组织均可见微弱荧光;三组心肌SERCA2a蛋白相对表达量显著高于肝肾(P<0.01),TVI组和IMI组心肌SERCA2a蛋白相对表达量明显高于IPI组(P<0.01);TVI组和IPI组心电图正常,IMI组2只大鼠发生室性早搏;三组转染AAV9-EGFP、AAV9-SERCA2a-EGFP与转染NS相比,心功能、组织病理学、血液生化学指标差异均无显著性(P>0.05)。结论三种方法转染滴度1×1011vg/mL AAV9-SERCA2a基因,TVI法和IMI法在心肌的有效性优于IPI法,TVI法和IPI法对机体的毒副作用小于IMI法,综合三种方法的有效性和毒副作用评价,TVI法优于IMI法和IPI法。

Objective To assess the efficiency and toxicity of adeno-associated virus serotype 9 （AAV9） mediated sareoplasmic reticulum Ca^2＋ ATPase 2a （SERCA2a） gene transfection to the myocardium in SD rats by different cardiac gene delivery methods. Methods The AAV9-SERCA2a-EGFP virus victor system was successfully constructed in vitro. According to the three cardiac gene delivery methods, 90 normal SD rats were randomly divided into three groups： tail vein injection （TVI） group, Intramyoeardial injeetion（IMI）group, and intraperieardial injection （IPI） group. Each group was transfected with AAV9-SERCA2a-EGFP, AAV9-EGFP, NS 200 μL at a titer of 1 × 10^11vg/mL using the appropriate method, respectively. At 30 days after the gene delivery, the expression of EGFP in the heart, liver and kidney tissues was determined by green fluorescence. Western blotting was performed to detect the relative expression levels of SERCA2a gene in the rat tissues. Surface 12-lead ECG was used to detect the incidence of arrhythmia. Histopathological changes were ohserved using HE staining. The changes of cardiac function were measured by echocardiography. Blood chemistry indicators were determined to assess the changes of liver and kidney functions. Results A large amount of green fluorescence could be observed in the left ventricular tissue in the three groups, but in the IMI group the observed green fluorescence was confined to the injection site. Faint green fluorescence was observed in the liver and kidney tissues of the three groups. The relative expression of SERCA2a protein in the myocardium of the three groups was significantly higher than that in the liver and kidney tissues （ P 〈 0.01 ）, and the myocardial relative expression of SERCA2a protein in the TVI and IMI groups was significantly higher than that in the IPI group （P 〈0.01 ）. ECG was normal in the TVI and IPI groups, but frequent premature ventricular beats were observed in two rats of the IMI group. There were no significant differences in the cardiac function, histopathology and blood chemistry indicators between the NS group and the three groups transfected with AAV9- SERCA2a-EGFP and AAV9-EGFP （ P 〉 O. 05 ）. Conclusions Transfection with a titer of 1 x 10^11 vg/mL AAV9-SERCA2a gene, the efficiency in the TVI and IMI groups is better than that in the IPI group, and the toxicity in the TVI and IPI goups is lower than that in the IMI group. Considering all the efficiency and toxicity caused by 1×10^11 vg/mL AAV9SERCA2a gene delivered with the three methods, the TVI method is better than the IMI and IPI methods.