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目的 探讨外源性 p1 4ARF基因表达于胰腺癌PC 3细胞后与另一重要细胞周期调控蛋白 p53表达水平的相互关系。 方法 将含人 p1 4ARFcDNA的重组质粒通过脂质体介导转染入胰腺癌PC 3细胞中 ,并筛选出阳性克隆 ,逆转录 聚合酶链反应 (RT PCR)、Westernblot法检测PC 3细胞转染前后p1 4ARF的mRNA及蛋白的表达 ,同时用Westernblot分析其内源性 p53蛋白表达水平的变化。噻唑蓝 (MTT)比色法测定PC 3细胞在转染前、后的生长曲线变化。结果 胰腺癌PC 3细胞中p1 4ARF基因缺失。外源性 p1 4ARF基因成功转染入PC 3细胞并获得 1 4× 1 0 3大小的p1 4蛋白表达 ,且内源性 p53蛋白表达水平在转染后明显增高。PC 3细胞在导入 p1 4ARF基因后第 1天及第 5天的生长抑制率分别为 2 2 %及 2 6 % ,两者差异有显著性 (P <0 .0 5)。结论 p1 4ARF基因可上调胰腺癌PC 3细胞的内源性p53蛋白表达水平 ,从而发挥其细胞周期阻滞功能。
Objective To investigate the relationship between the expression of exogenous p14ARF gene and the expression of another important cell cycle regulatory protein p53 in pancreatic cancer PC-3 cells. Methods Recombinant plasmids containing human p1 4ARF cDNA were transfected into PC3 cells by lipofectamine. The positive clones were screened by reverse transcriptase polymerase chain reaction (RT PCR). The transfected PC3 cells were detected by Western blot Before and after p1 4ARF mRNA and protein expression, while using Westernblot analysis of endogenous p53 protein expression levels. The growth curves of PC 3 cells before and after transfection were measured by MTT assay. Results The p1 4ARF gene was deleted in PC3 cells. The exogenous p1 4ARF gene was successfully transfected into PC 3 cells and the expression of p1 4 protein of 14 × 10 3 was obtained. The expression of endogenous p53 protein was significantly increased after transfection. The growth inhibition rates of PC 3 cells on day 1 and day 5 after introduction of p1 4ARF gene were 22% and 26%, respectively, with significant difference (P <0.05). Conclusion p1 4ARF gene can up-regulate the expression of endogenous p53 protein in PC-3 cells of pancreatic cancer and thus exert its cell-cycle arrest function.