佛波酯对人胃癌细胞Ha-ras基因启动子及启动子结合蛋白的影响
Acta Anatomica Sinica（2006）
Abstract
目的探讨佛波酯(PMA)对人胃癌细胞BGC-823 Ha-ras基因启动子活性及启动子结合蛋白活性的影响.方法通过RT-PCR和运用自行构建的真核表达载体prasEYFP转染细胞进行荧光细胞检测,并应用凝胶电泳移动检测、Southwestern blotting等方法检测PMA(100 μg/L)处理BGC-823细胞72和96 h后,对Ha-ras基因表达水平、Ha-ras基因启动子活性以及对Ha-ras启动子结合蛋白的影响. 结果经PMA(100 μg/L)处理72和96 h,与未处理细胞相比,BGC-823细胞中Ha-ras基因表达显著降低,Ha-ras基因启动子活性分别被抑制65.2%和71.8%;同时两个Ha-ras启动子结合蛋白活性也显著降低,经检测,其分子量分别约为18 kD,35 kD. 结论PMA长期处理通过降低人胃癌细胞中Ha-ras启动子结合蛋白活性使Ha-ras启动子活性被抑制,从而在转录水平上调节Ha-ras基因表达.
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Key words
Gel mobility shift assay,Ha-ras gene,Ha-ras promoter and promoter-binding proteins,Human gastric carcinomal cells (BGC-823),PMA,Southwestern blotting
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