中文摘要：

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。

英文摘要：

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.