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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S、outhwestern blotting等方法检测PMA(100μg/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(100μg/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。
Objective To investigate the effect of phorbol ester (PMA) on the promoter activity and promoter-binding protein activity of Ha-ras gene in human gastric cancer cell line BGC-823. Methods Fluorescent cells were detected by RT-PCR and transfected into prasEYFP by using self-constructed eukaryotic expression vector. Cell proliferation was detected by gel electrophoresis, Western blotting and Western blotting, respectively. The proliferation of BGC-823 cells treated with PMA (100μg / L) After 96 h, the expression of Ha-ras gene, Ha-ras gene promoter activity and Ha-ras promoter binding protein were studied. Results Compared with untreated cells, the expression of Ha-ras gene in BGC-823 cells was significantly decreased and the promoter activity of Ha-ras gene was inhibited by 65.2% and 71.8% at PMA (100μg / L) for 72 and 96 h, respectively. At the same time, the activity of the two Ha-ras promoter-binding proteins was also significantly reduced. The molecular weights of the two Ha-ras promoters were about 18 kD and 35 kD, respectively. Conclusion Long-term treatment of PMA can regulate the expression of Ha-ras gene at the transcriptional level by inhibiting the activity of Ha-ras promoter by reducing the activity of Ha-ras promoter-binding protein in human gastric cancer cells.