细胞周期素D_2启动子曲古菌素A效应区域的研究  

Determination of cyclin D_2 promoter region in response to trichostatin A treatment

作　　者：郭志义[1] 郝小惠[1] 田炜[1] 谭菲菲[2] 姜雪莲[2] 李潇婷 胡倩倩[2] 杨方[1] 

机构地区：[1]河北联合大学医学实验研究中心,河北省煤矿卫生与安全重点实验室,河北唐山063000 [2]河北联合大学生命科学院,,河北唐063000

出　　处：《第三军医大学学报》2011年第14期1458-1461,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(31050010);河北省教育厅青年基金(2010154);华北煤炭医学院硕博启动基金(BS06002)~~

摘　　要：目的研究在曲古菌素A(trichostatin A,TSA)处理条件下细胞周期素D2的调控机制。方法细胞周期素D2的mRNA水平通过RT-PCR方法检测。细胞周期素D2的启动子片段以A549细胞基因组DNA为模板,通过高保真PCR扩增获得,并克隆到pGL3-BASIC载体中;并以此为基础通过高保真PCR扩增出不同长度的启动子片段。启动子相对活性通过萤火虫酶报告基因方法确定。结果在TSA处理条件下,细胞周期素D2的mRNA水平上升。共构建4个以不同长度细胞周期素D2启动子片段驱动的报告基因质粒,分别命名为pGL3-1816CD2-LUC、pGL3-1358CD2-LUC、pGL3-720CD2-LUC和pGL3-524CD2-LUC。在TSA处理条件下,启动子活性的变化在-524 bp删切质粒时消失。结论 TSA条件下细胞周期素D2 mRNA水平上升是启动子依赖的;细胞周期素D2启动子的TSA效应区域在-720～-524 bp之间。Objective To explore the regulation mechanism of cyclin D2 under trichostatin A（TSA） treatment.Methods The mRNA level of cyclin D2 was detected by RT-PCR.Cyclin D2 promoter fragment was amplified by high-fidelity PCR with A549 cell genome DNA as a template,and was then inserted into plasmid vector pGL3-BASIC.Truncated promoter fragments were amplified based on the recombinant plasmid.The relative activity of the promoter fragments was evaluated with the firefly luciferase reporter gene method.Results The mRNA level of cyclin D2 increased under TSA treatment.Four plasmids carrying the truncated cyclin D2 promoter fragments to control reporter gene expression were constructed and named pGL3-1816CD2-LUC,pGL3-1358CD2-LUC,pGL3-720CD2-LUC and pGL3-524CD2-LUC.Under TSA treatment,the promoter activity diminished when the promoter fragment was truncated to 524 bp.Conclusion The mRNA level increase of cyclin D2 depends on the promoter under TSA treatment.The TSA response region in cyclin D2 promoter is located in the range from 720 bp to 524 bp.

关 键 词：细胞周期素D2 启动子 报告基因 曲古菌素A 

分 类 号：R979.1[医药卫生—药品] R966[医药卫生—药学]