| 徐晓霞,周祁娜,姜涛,王红丽,王欢,季萌,王凌鹏,张广伟,侯月梅.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[J].中国实验动物学报,2013,21(3):. |
| 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 transfected by different cardiac gene delivery methods at the same titer in rats |
| 投稿时间：2012-09-05 修订日期：2012-10-09 |
| DOI： |
| 中文关键词: 转基因 有效 毒副作用 |
| 英文关键词:Gene delivery Efficiency Toxicity |
| 基金项目:国家自然科学基金项目（面上项目，重点项目，重大项目） |
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| 中文摘要: |
| 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 reticulum Ca2+ATPase 2a,SERCA2a)基因的9型腺相关病毒(adeno-associated virus serotype9,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1×1011 vg/mL AAV9-SERCA2a-EGFP、AAV9-EGFP、NS各200µL。转染后30d，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SERCA2a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、血液生化学指标均无显著差异（P>0.05）。结论 三种方法转染滴度1×1011 vg/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。 |
| 英文摘要: |
| Objective To assess the efficiency and toxicity of adeno-associated virus serotype 9 (AAV9) mediated sarcoplasmic reticulum Ca2+ATPase 2a (SERCA2a) gene transferring to myocardium in SD rats by different cardiac gene delivery methods. Methods The AAV9-SERCA2a-EGFP virus victor system was constructed in vitro successfully. According to the three cardiac gene delivery methods, 90 normal SD rats were randomly divided into three groups: TVI (Tail vein injection，TVI)group, IMI（Intramyocardial injection，IMI）group, and IPI (Intrapericardial injection，IPI)group. Each group was respectively transferred with AAV9-SERCA2a-EGFP, AAV9-EGFP, NS 200µL at the titer of 1×1011 vg/mL using the appropriate method. After 30 days of gene delivery, the expression of EGFP in the tissue of heart, liver and kidney was observed by inverted fluorescence microscopy. Western blotting was performed to detect relative expression levels of SERCA2a gene in rats' tissue. Surface 12-lead ECG was used to record the incidence of arrhythmia. HE staining observed the histopathological changes. The changes of cardiac function was measured by echocardiogram. Blood chemistry indicators were used to assess the changes in liver and kidney. Results Left ventricular could observed a large number of green fluorescent in three groups and IMI group of green fluorescent confined in the injection point, liver and kidney were seen faint green fluorescence in three groups; The relative expression of SERCA2a protein of myocardium in the three groups was significantly higher than liver and kidney (P<0.01), and the myocardial relative expression of SERCA2a protein in TVI group and IMI group was significantly higher than IPI group (P<0.01). ECG was normal in TVI group and IPI group, but frequent premature ventricular beats were observed in two rats of IMI group.There were no significant difference in cardiac function, histopathology and blood chemistry indicators between transferring AAV9-SERCA2a-EGFP, AAV9-EGFP and transferring NS (P>0.05). Conclusion Transfected with a titer of 1×1011 vg/mL AAV9-SERCA2a gene, the efficiency in TVI group and IMI goup was better than IPI goup, and the toxicity in TVI group and IPI goup was lower than IMI group. Considering the evaluation of efficiency and toxicity by 1×1011 vg/mL AAV9-SERCA2a gene delivering in three methods, the TVI method is better than IMI and IPI method. |
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