rAAV_2VEGF_(165)与rAAV_2ANG-1联合转染促猪缺血心肌血管生成的研究  

Angiogenesis in chronic ischemic porcine myocardium after transfer of VEGF_(165) and angiopoietin-1 mediated by recombinant adeno-associated viral vector

作　　者：朱成楚[1] 陈仕林[2] 刘玉清 唐礼江[1] 林仙方[1] 包卫光[1] 马德华[1] 朱广球[1] 周文娟[1] 周仪林[1] 杜丛文[1] 

机构地区：[1]浙江省台州医院心胸外科,浙江临海317000 [2]南京医科大学附属江苏省肿瘤医院胸外科,江苏南京210004 [3]阜外医院临床心血管药理中心,北京100037

出　　处：《浙江大学学报（医学版）》2010年第6期610-617,共8页Journal of Zhejiang University(Medical Sciences)

基　　金：国家人事部博士后基金资助课题(2002032188);浙江省卫生厅重点资助项目(2005ZD012)

摘　　要：目的:探讨联合应用血管内皮细胞生长因子(VEGF165)及血管生成素(ANG-1),对猪慢性缺血心肌中血管生成效应及心功能改善的影响。方法:完全腔镜下放置血管缩窄环于小型猪左冠状动脉回旋支(LCX),建立慢性心肌缺血模型。6周后行心电图、冠状动脉造影和心脏超声检查,确认LCX闭塞或相应心肌的缺血。16只动物随机均分为:单纯注射转染VEGF165组(组Ⅰ,n=4)、单纯注射转染ANG-1组(组Ⅱ,n=4)、联合注射转染VEGF165与ANG-1组(组Ⅲ,n=4)和注射磷酸盐缓冲液PBS对照组(组Ⅳ,n=4)。各组在胸腔镜下,心肌内直接注射rAAV2ANG-1、rAAV2VEGF165,剂量为5×1011vg(virus/genome)。治疗后,不同时间点ELISA检测VEGF165和ANG-1蛋白的分泌,冠状动脉造影观察侧枝循环形成的情况,行心脏超声检查观察左心室功能变化。3个月后取注射部位心肌,用Western blot检测VEGF165和ANG-1蛋白的表达情况,免疫组织化学观察心肌毛细血管密度与微小动脉生成情况。结果:放置血管缩窄环后6周,所有动物均出现LCX完全/次全闭塞,或LCX支配区域的心肌缺血。Ⅰ、Ⅲ两组猪体内的VEGF165蛋白分泌水平从基因转染后第7天开始上升,并于第14天达到高峰,随后逐渐下降,尤以Ⅰ组分泌水平为高;其它两组各时间点及组间的VEGF165蛋白分泌水平差异无统计学意义(P>0.05)。同样,Ⅱ、Ⅲ两组猪体内的ANG-1蛋白分泌水平的变化与VEGF165蛋白分泌水平类似,尤以Ⅱ组分泌水平为高(P<0.01);其它两组各时间点及组间的ANG-1蛋白分泌水平差异无统计学意义;组Ⅲ中VEGF165与ANG-1蛋白水平变化同步。Western blot检测显示,基因治疗后3个月,组ⅢVEGF165、ANG-1蛋白水平都显著高于组Ⅳ(P<0.01),组Ⅲ与组Ⅰ间VEGF165蛋白表达无显著差异(P>0.05),组Ⅲ与组Ⅱ间ANG-1蛋白表达差异无统计学意义(P>0.05)。基因治疗3个月后,组Ⅲ左心室室腔缩小,心肌收缩力增强,LCX及其分支较基因转染前增�Objective: To study the effects of combination of angiopoietin-1(ANG-1) and vascular endothelial growth factor165(VEGF165) gene transfer mediated by recombinant adeno-associated viral vector on the neovascularization in chronic ischemic porcine myocardium.Methods: An ameroid constrictor was implanted around the left circumflex coronary artery(LCX) via endoscopy.Six weeks later,coronary angiography revealed that the myocardial ischemia was established by gradual occlusion of the left circumflex coronary artery(LCX).Sixteen swine with the total occlusion or partial stenosis(>85%) of the LCX were divided into 4 groups(4 in each group):group Ⅰ,group Ⅱ and group Ⅳ(control) received direct myocardium injection of rAAV2VEGF165,rAAV2ANG-1 or PBS alone,respectively;group Ⅲ received rAAV2VEGF165 and rAAV2ANG-1.Selective coronary angiography and ultrasonography were performed perioperatively to evaluate the cardiac function and the formation of collateral circulation.The expression of VEGF165 and ANG-1 proteins were assessed using ELISA or Western blot.The degree of angiogenesis was assessed by use of immunohistochemical analysis.Results: Angiography showed that the occlusion of all LCX was completed or exceeded 95% 6 weeks after ameroid constrictor implantation,indicating the successful establishment of animal model.The expression levels of VEGF165 in group Ⅰ and Ⅲ and ANG-1 in groups Ⅱ and Ⅲ began to increase at d7 after transfection and reached the peak at d14;then decreased gradually to the normal level after 3 months.The expression levels of VEGF165 in group Ⅱ and group Ⅳ or that of ANG-1 protein in group Ⅰ and group Ⅳ had no markedly changes at different time after transfection.There were significant increase in capillary density and arteriole density and more side branch vessels formed in group Ⅲ compared with other groups.Echocardiographic measurements showed that the left ventricular systolic function of animals in groups Ⅰ,Ⅱ and Ⅲ increased significantly after gene transfection,espe

关 键 词：心肌缺血/病理学 血管生成素1/遗传学 慢性心肌缺血 腺相关病毒 血管形成素-1 血管内皮细胞生长因子类/遗传学 血管新生 

分 类 号：R541.4[医药卫生—心血管疾病]