构建表达Livin的逆转录病毒载体及其在大鼠心肌细胞中的表达

Construction of retroviral vector expressing Livin and its expression in cardiomyocytes of rats

目的构建表达Livin的逆转录病毒载体,并观察其转染大鼠心肌细胞后,心肌细胞Livin表达水平的变化情况。方法①建立表达Livin的逆转录病毒载体,并用PCR酶切及DNA测序鉴定;②健康SD大鼠30只,分为正常组、Livin组和空载体对照组;Livin组开胸心肌内注射表达Livin的逆转录病毒载体;空载体组开胸注射空载体,两组均于手术24 h后再开胸取出心脏;正常组直接开胸游离心脏;③荧光定量PCR检测大鼠心肌LivinmRNA表达量。结果①成功构建了表达Livin的逆转录病毒载体;②荧光定量PCR检测到正常大鼠心肌细胞有Livin表达;③注射表达Livin的逆转病毒载体后Livin表达显著增多。结论表达Livin的逆转录病毒载体可以在大鼠心肌内成功表达,转染表达Livin的逆转录病毒载体,有可能成为增加大鼠心肌抗凋亡基因、抑制心肌细胞凋亡、防治再灌注损伤的有效治疗措施。

Objective To observe the livin protein expression in rat cardiomyocytes after the transfection of retroviral vectors expressing Livin.Methods ① Livin gene from mice MA782 cell was amplificated by reverse-transcription polymerase chain reaction（RT-PCR）and first cloned into pGEM-T vector,then digested by XhoⅠ/HindⅢ and identified by PCR,and then subcloned into the retroviral vector pLNCX2.pLNCX2-Livin was transferred into packaging cell PT67 after XhoⅠ/HindⅢ digestion and identified by PCR.PT67/pLNCX2-Livin cells were selected.A pure packaging cell line/pLNCX2-Livin cell expressing Livin was cultured.The virus titer was assayed.②Thirty rats were divided into normal group,contrl group and Livin group.Retroviral vector expressing Livin was injected into rat myocardium in the Livin group.Retroviral vector without Livin gene was injected into rat myocardium in the control group and the heart was removed 24 h later.③ The Livin mRNA was determined by a real time PCR.Results ①A 816 bp Livin cDNA was obtained by RT-PCR amplification.The recombinant vector pLNCX2-Livin containing Livin gene was confirmed.The virus titer of the pure packaging cell line/PT 67/pLNCX2-Livin was 3.9×10^7 IU/ml.② The Livin mRNA was positive in normal rats and increased significantly after the transfection with retroviral vector expressing Livin.Conc-lusion The retroviral vectors are established and transfected into rat myocardium successfully.Transfecting of retroviral vector expressing Livin into rat myocardium could be useful for treatment of ischemia reperfusion injury.