人PI3KCG慢病毒载体的构建及其在乳大鼠原代心肌细胞中的表达和初步功能检测

目的:构建含有人磷脂酰肌醇3-激酶p110γ(phosphatidylinositol 3-kinase,catalytic subunit gamma,PI3KCG)基因的慢病毒载体,鉴定其在乳大鼠原代心肌细胞中的表达并作初步功能检测。方法:利用同源重组的方法构建含PI3KCG基因慢病毒载体质粒,测序鉴定后采用脂质体转染法同慢病毒系统三质粒共转染293T细胞,转染后24 h和48 h荧光显微镜下观察阳性对照组中的绿色荧光蛋白的表达,同时PCR法鉴定重组PI3KCG慢病毒质粒转染293T细胞成功,收集72 h病毒上清。培养乳大鼠原代心肌细胞,分为对照组,缺氧/复氧组,空载体阳性对照组,PI3KCG实验组,PI3KCG+Ly294002组5组,将含PI3KCG慢病毒载体的病毒液转染心肌细胞,检测各组的细胞培养上清中的乳酸脱氢酶浓度,观察预转染PI3KCG基因对心肌细胞缺氧/复氧过程中的保护作用。结果:成功构建含有PI3KCG基因的慢病毒载体质粒,并在293T细胞中成功表达。与缺氧/复氧组比较,PI3KCG组心肌细胞培养液中乳酸脱氢酶水平明显降低(P<0.01);心肌细胞的存活率、搏动频率明显升高(P<0.01)。结论:PI3KCG慢病毒载体有望成为探讨PI3K/AKT信号通路激活在防止心肌细胞缺血再灌注损伤的有效工具。

Construction of recombinant lentiviral vector of PI3KCG,identification of its expression in neonatal rat cardiomyocytes and prelimary function test

Objective:To construct lentiviral expression vector contained phosphatidylinositol 3-kinase p110 gamma(PI3KCG), and identify its expression in neonatal rat cardiomyocytes and detect its prelimary function. Methods:The PI3KCG lentiviral vector plasmid (PLV-PI3KCG)was constructed by homologous recombination method. The three plasmids of PLV-PI3KC and of lentivirus system were co-transfected into human embryonic kidney 293T cells by using lipofectamine. The expression of green fluorescent protein (GFP)in the control group was examined by using fluorescent microscope at 24 h and 48 h after transfection. Recombinant PLV-PI3KCG plasmid was successfully identified in 293T cells detected by polymerase chain reaction (PCR). The viral supernatant was collected with 72 h after transfection. The ischemia/reoxygenation (I/R)injury model of neonatal rat myocardial cells was established. Myocardial cells isolated from SD neonatal rats were random division into five groups after being cultured for 3d: the normal control group, the I/R group, the null vector positive control group, the PI3KCG transfection preconditioning group and the PI3KCG transfection+ Ly294002 group. Various techniques were adopted to detect the products of cells and cellular:the cardiomycytes beat frequency, the levels of myocardial cells viability rate and the levels of lactate dehydrogenase (LDH). Results:The PLV-PI3KCG plasmid was constructed and expressed in the 293T cells successfully. Compared with the I/R group, the myocardial cells viability and cardiomycytes beat frequency of the PI3KCG transfection preconditioning group were significantly increased (P < 0.01,respectively), and released LDH were significantly decreased (P < 0.01,respectively). Conclusion:PLV-PI3KCG plasmid was expected to become an available vector to investigate PI3K/Akt pathway in the cardiocytes ischemia reperfusion injury process.