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  

EFFECT OF PMA ON THE ACTIVITY OF Ha-ras PROMOTER AND PROMOTER BINDING PROTEIN IN HUMAN GASTRIC CARCINOMA CELLS

作　　者：高萍[1] 王永军[1] 柳惠图[1] 

机构地区：[1]北京师范大学生命科学学院细胞增殖与调控生物学教育部重点实验室,北京100875

出　　处：《解剖学报》2006年第1期62-65,共4页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(39870366)

摘　　要：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 study the effect of PMA on the activity of Ha-ras promoter and promoter-binding protein in BGC-823 cells. Methods Human gastric carcinoma cells（BGC-823） were treated with PMA（100 μg/L） for 72 hours and 96 hours respectively. By using tripure isolation reagent the RNA of treated and untreated cells were extracted according to the procedure of the manufacturer. The expression of Ha-ras gene was detected by reversed transcriptase polymerase chain reaction. The recombinant plasmid pRasEYFP which contained the regulation fragment of Ha-ras and EYFP gene was constructed. Ha-ras regulation fragment was located upstream EYFP gene. The recombinant plasmid pRasEYFP was transiently transfected to BGC-823 eells. Using the EYFP as reporter, the activity of Ha-ras promoter was assayed. The Ha-ras promoter-binding proteins were examined by gel mobility shift assay and Southwestern blotting analysis. Results Compared with untreated cells the level of Ha- ras mRNA was decreased markedly and the activity of promoter of Ha-ras was inhibited by 65.2% and 71.8% in BGC-823 cells after treatment with PMA（ 100μg/L） for 72 hours and 96 hours respectively. The activity of two binding proteins with Ha-ras promoter was decreased obviously in BGC-823 cells treated by PMA for 72 hours and 96 hours. Molecular weights of the two proteins were about 18 kD, 35 kD respectively. Conclusion PMA playsan important role in regulation of expression of Ha-ras gene at transeriptional level by effect the activity of Ha-ras promoter and promoter-binding proteins in human gastric carcinoma cells（ BGC-823 ）.

关 键 词：佛波酯 Ha—ras基因 Ha—ras基因启动子与结合蛋白 人胃癌细胞BGC-823 凝胶电泳移动检测 DNA免疫印迹法 

分 类 号：Q253[生物学—细胞生物学]