血管生成素-1基因治疗兔心肌梗塞的实验观察

Effect of Angiopoietin-1Gene Transfer Mediated by Recombinant Adenovirus on Neoangiogenesis in Ischemic Myocardium

探讨血管生成素_1(Ang1)基因经重组腺病毒介导转移促进心肌缺血区血管形成的作用 .应用共转染 2 93细胞 ,制备纯化Ang1重组腺病毒及LacZ重组腺病毒 .将Ang1重组腺病毒及LacZ重组腺病毒直接注射感染兔冠状动脉结扎后缺血心肌细胞 ,分别应用RT_PCR方法及X_gal染色测定外源基因在转导心肌中的表达 ,用免疫组化及冠状动脉造影方法观察缺血心肌内血管生长及侧支循环形成的情况 ,并用心脏超声检查观测了注射Ang1重组腺病毒后心功能变化 .结果显示重组腺病毒直接注射感染兔缺血心肌后能有效表达目的基因 ,X_gal染色显示LacZ基因转移后第 3、7、14、2 8d注射部位心肌均见蓝染 ,尤以 7d明显 ,RT_PCR测定提示Ang1基因在转移后第 3d心肌中即有表达 ,7、14d仍呈阳性表达 .转移 2 8d后新生毛细血管密度及侧支循环形成明显高于对照组 (P <0 .0 1) .心肌梗塞发生后各组的心功能随时间延长均有所改善但 14d内各组间无明显差异 ,2 8d后Ang1组改善幅度明显高于对照组 (P <0 .0 1) .本实验表明腺病毒介导的Ang1基因在兔缺血心肌中能有效表达 ,促进新生血管形成 ,改善心脏功能 .

To product the angiopoietin-1recombinant adenovirus and study the potential effects of angiopoietin-1via adenovirus mediated gene transfer on ischemic myocardium of rabbits.The replication-deficient adenovirus was generated by the cotransfection of293cell and the homologous recombination between the cosmid DNA of the Ang1and Ad5-TPC.60Male New Zealand White rabbits underwent high positioned double-ligation of the anterior descending left coronary artery,and were divided into three groups;Ang1group received direct myocardium injection of Ang1recombinant adenovirus.LacZ group and DMEM group received only LacZ recombinant adenovirus or DMEM as control respectively.To observe the efficiency and stability of adenovirus- mediated gene transfer into rabbit myocardium,additional8rabbits received direct myocardium injection of LacZ recombinant adenovirus.The expression LacZ or Ang1gene was investigated with X-gal staining or RT-PCR respectively,the degree of neovascularization in the ischemic myocardium was compared at different time after infection using angiography and immunohistochemical analysis.The echocardiographic changes were measured on the7th、14th、28th postoperative day.X-gal staining of section from hearts infected with Adv5-CAG/LacZ showed blue staining and RT-PCR revealed that the Ang1gene could be transcripted in vivo,The high level of gene expression only persisted about one month.Angiography and immunohistochemical analysis demonstrated that the transferred Ang1gene could promote the formation of new blood vessels in the region of ischemical myocardium.Echocardiographic measurements presented that animals in three groups had no significantly difference in the left ventricular systolic function before operation and at the7th and14th days,but the left ventricular systolic function in Ang1group was better than that in DMEM group or LacZ group at the28th day(P<0.01).Adv5-vectors provide an efficient method for in vivo expression in myocardium.Angiopoietin-1recombinant adenovirus was of the ability to promote the angiogenesis and improve the left ventricular systolic function in the rabbit model of myocardium infarct.