【摘 要】
：
目的：评价该系统结合小干扰RNA(siRNA)对大鼠肝癌细胞乏氧条件下乏氧诱导因子1α(HIF-1α)基因表达的干扰效果。方法：CoCl2建立细胞乏氧模型,设计并筛选HIF-1α siRNA.使用CDAN脂质体、siRNA、碘油、碘帕醇制备基因转染系统,荧光显微镜及流式细胞术检测转染系统稳定性和转染效率。使用转染系统结合筛选的HIF-1α siRNA干扰乏氧条件下CBRH-7919细胞,Real-
【机 构】
：
复旦大学附属肿瘤医院放射诊断科 上海200032
论文部分内容阅读
目的：评价该系统结合小干扰RNA(siRNA)对大鼠肝癌细胞乏氧条件下乏氧诱导因子1α(HIF-1α)基因表达的干扰效果。方法：CoCl2建立细胞乏氧模型,设计并筛选HIF-1α siRNA.使用CDAN脂质体、siRNA、碘油、碘帕醇制备基因转染系统,荧光显微镜及流式细胞术检测转染系统稳定性和转染效率。使用转染系统结合筛选的HIF-1α siRNA干扰乏氧条件下CBRH-7919细胞,Real-TimePCR检测HIF-1α基因表达水平。多组数据比较使用单因素方差分析，P<0.05为检验标准。结果：200 uM CoC12模拟乏氧24小时使HIF-1α，血管内皮生长因子，基质金属蛋白酶2基因表达分别上调2.27±0.09,5.29±0.08,2.75±0.09倍。通过筛选，得到2条序列 siRNAl,siRNA3分别使HIF-1α基因表达降低至0.18±0.08,0.22±0.03。使用N/P为1:1,2:1,4:1CDAN/siRNA复合物与体积比为2％碘油、30％碘帕醇通过常规“泵法”及涡旋法可制备具有一定稳定性的基因转染系统，N/P为1:1,2:1,4:1时细胞转染效率分别为49.7％±4.66,45.5％±5.11,20.4％±3.28;N/P为1:1,2:1时siRNAl对HIF-1α基因表达降低至0.42±0.09,0.65±0.03;siRNA3为0.52±0.07,0.63±0.06。结论：使用CDAN,siRNA,碘油、碘帕醇可以建立具有一定体外转染效率的稳定基因转染系统，结合siRNA对乏氧条件下大鼠肝癌细胞HIF-1α表达具有一定沉默效果。
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