rAAV9介导的siRNA敲减p65对压力超负荷大鼠心肌纤维化和凋亡的影响  

Effects of rAAV9-mediated siRNA for p65 knockdown on myocardial fi⁃brosis and apoptosis in rats with pressure overload

作　　者：王栩越 包雅丽 张甜 凌灿[1] 王一平 吴振宏 孙湛[1] WANG Xu-yue;BAO Ya-li;ZHANG Tian;LING Chan;WANG Yi-ping;WU Zhenhong;SUN Zhan(Basic Medical College of Xinjiang Medical University,Urumqi 830011,China)

机构地区：[1]新疆医科大学基础医学院,新疆乌鲁木齐830011

出　　处：《中国病理生理杂志》2020年第11期1938-1944,共7页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.81560078);新疆维吾尔自治区自然科学基金资助项目(No.2019D01C207)。

摘　　要：目的:探讨重组腺相关病毒血清型9(rAAV9)介导的敲减p65基因表达对压力超负荷大鼠心功能的作用及可能机制。方法:通过腹主动脉缩窄手术(AAC)构造大鼠左心室肥厚模型。将造模的SD大鼠随机分为假手术组、AAC组、AAC+rAAV9-eGFP组和AAC+rAAV9-eGFP-P65-siRNA组。假手术组大鼠开腹后直接关闭腹腔;其余3组大鼠开腹结扎腹主动脉后关闭腹腔,术后3 d对AAC大鼠分别尾静脉注射生理盐水、rAAV9-eGFP和rAAV9-eGFP-P65-siRNA。4周后,测定血流动力学相关指标;计算心脏质量参数;Masson染色检测心肌纤维化程度;TUNEL染色观察细胞凋亡水平;Western blot检测心肌P65的表达水平;ELISA检测各组大鼠血清肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)的含量。结果:AAC组和AAC+rAAV9-eGFP组中P65表达量较假手术组上升(P<0.05);AAC+rAAV9-eGFP-P65-siRNA组中P65表达量较AAC组明显降低(P<0.05)。AAC组和AAC+rAAV9-eGFP组大鼠收缩压、舒张压、左室重量/体重、心肌细胞凋亡率及TNF-α和IL-6水平均较假手术组增高(P<0.05);与AAC组相比,AAC+rAAV9-eGFP-P65-siRNA组大鼠上述指标均下降(P<0.05)。Masson染色结果显示,与假手术组相比,AAC组和AAC+rAAV9-eGFP组大鼠心肌组织中胶原蛋白的沉积增多,而rAAV9-eGFP-P65-siRNA可以缓解高血压诱导的AAC大鼠心肌纤维化发生。结论:敲减p65基因表达可减轻压力超负荷大鼠左心室纤维化和细胞凋亡程度,其机制与调节NF-κB通路,减少炎症反应有关。AIM:To investigate the effect of p65 gene knock-down mediated by recombinant adeno-associated virus serotype 9(rAAV9)on the cardiac function of pressure overload rat and its possible mechanism.METHODS:The rat model of left ventricular hypertrophy was established by abdominal aortic coarctation(AAC).SD rats were randomly di⁃vided into sham operation group,AAC group,AAC+rAAV9-eGFP group and AAC+rAAV9-eGFP-P65-siRNA group.The abdominal cavity was closed directly after laparotomy in the rats of sham operation group,the abdominal cavity was closed after ligation of the abdominal aorta in the rats of AAC group,and normal saline,rAAV9-eGFP and rAAV9-eGFP-P65-siRNA were injected into the tail vein 3 d after operation.After 4 weeks,the hemodynamic indexes were measured,the heart mass parameters were calculated,the degree of myocardial fibrosis was detected by Masson staining,the expression level of myocardial P65 was detected by Western blot,the degree of apoptosis was detected by TUNEL staining,and the serum contents of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)of the rats in each group were measured by ELISA.RESULTS:The expression of P65 in AAC group and AAC+rAAV9-eGFP group was higher than that in sham op⁃eration group,while the expression of P65 in AAC+rAAV9-eGFP-P65-siRNA group was significantly lower than that in AAC group.The levels of systolic blood prossure(SBP),diastolic blood pressure(DBP),left ventricular weight/body weight(LVW/BW),cardiomyocyte apoptotic rate and TNF-αand IL-6 in AAC group and AAC+rAAV9-eGFP group were higher than those in sham operation group,while SBP,DBP,LVW/BW,cardiomyocyte apoptosis rate and TNF-αin AAC+rAAV9-eGFP-P65-siRNA group were significantly lower than those in AAC group.The results of Masson staining showed that the deposition of collagen in cardiac tissue in AAC group and AAC+rAAV9-eGFP group was higher than that in sham operation group,and treatment with rAAV9-eGFP-P65-siRNA alleviated hypertension-induced fibrosis.CON⁃CLUSION:Knockdown of p65 gene reduces

关 键 词：重组腺相关病毒血清型9 P65蛋白 高血压 心肌纤维化 细胞凋亡 

分 类 号：R542.23[医药卫生—心血管疾病] R363.2[医药卫生—内科学]