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目的:研究佛波醇酯(PMA)对HeLa细胞Ha-ras基因启动子活性的影响.方法:利用MTT法检测PMA对HeLa细胞生长速率的影响,并通过逆转录聚合酶链反应法测定HeLa细胞Ha-ras基因表达水平,进一步将Ha-ras基因上游调控序列插入到不含启动子元件的EYFP基因的上游,构建了重组质粒pRasEYFP,并将其瞬时转染到heLa细胞中.以EYFP,作为报告基因分析了PMA长期处理对Ha-ras基因启动子活性的影响.在此基础上对PMA处理的HeLa细胞中蛋白激酶C(PKC)的活性进行了检测.结果:与未处理细胞相比,PMA长期处理引起HeLa细胞生长速率明显降低.经PMA处理(100μg/L)48小时和72小时,与未处理细胞相比HeLa细胞中Ha-ras基因的表达降低,Ha-ras基因启动子活性分别下降了34.0％和26.7％,同时PKC的活性分别降低了76.3％和73.2％.结论:PMA在Ha-ras基因启动子活性的调节中扮演着重要的角色.其作用的机理可能与PKC信号通路有关.
Objective: To investigate the effect of phorbol ester (PMA) on the promoter activity of Ha-ras gene in HeLa cells.Methods: The effect of PMA on the growth of HeLa cells was detected by MTT assay and HeLa cells were determined by reverse transcription polymerase chain reaction Ha-ras gene expression levels, and further upstream of Ha-ras gene regulatory sequence inserted into the promoter element EYFP gene upstream to construct a recombinant plasmid pRasEYFP, and transiently transfected into heLa cells with EYFP, as Reporter gene analysis of the PMA long-term treatment of Ha-ras gene promoter activity in the PMA-treated HeLa cells in the protein kinase C (PKC) activity were measured.Results: Compared with untreated cells, The long-term treatment of PMA caused HeLa cell growth rate was significantly reduced.PHA treatment (100μg / L) 48 hours and 72 hours, compared with untreated cells HeLa cells Ha-ras gene expression decreased Ha-ras gene promoter activity Decreased by 34.0% and 26.7%, respectively, and the activity of PKC decreased by 76.3% and 73.2% respectively.Conclusion: PMA plays an important role in the regulation of Ha-ras gene promoter activity.The mechanism may be related to PKC signaling pathway related.