细胞周期素d2启动子trichostatina效应区域的研究

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1、细胞周期素D2启动子TrichostatinA效应区域的研究郭志义1，郝小惠1，田炜1，谭菲菲2，姜雪莲2，李潇婷2，胡倩倩2，杨方1(063000河北唐山，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1，生命科学院)[摘要]目的研究在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为模版，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

2、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删切质粒时消失。结论TSA条件下细胞周期素D2mRNA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到-524之间。[关键词]细胞周期素D2；启动子；报告基因；Trichos

3、tatinA[中图法分类号]Q78[文献标志码]ADeterminationofcyclinD2promoterregioninresponsetoTrichostatinAtreatmentGuoZhiyi1，HaoXiaohui2，TanFeifei,2JiangXuelian2,YangFang1（1MedicalExperimentalandResearchcenter,HebeiUnitedUniversity,HebeiProvinceKeyLaboratoryofOccupationalHealthandSafetyforC

4、oalIndustry，China0630002SchoolofLifeScience,HebeiUnitedUniversity,China063000）［Abstract］ObjectivetoexplorethecyclinD2expressionunderTrichostatinA(TSA)treatmentinA549cellline.MethodscyclinD2mRNAlevelwasdetectedbyRT-PCR.HumancyclinD2promoterfragmentwasamplifiedbyHigh-Fideli

5、tyPCRwiththegenomeDNAastemplateisolatedfromA549cells,andtheninsertedintopGL3-BASICvector.ThetruncationseriesofhumancyclinD2promoterfragmentwereamplifiedbasedontheplasmid.Therelativepromoteractivitywasevaluatedbyreportergeneanalysis.ResultscyclinD2mRNAlevelincreasedunderTS

6、Atreatment.Fourtruncatedplasmidswereconstructedwithnameassigned:pGL3-1816CD2-luc、pGL3-1358CD2-luc、pGL3-720CD2-lucandpGL3-524CD2-luc.ThechangeofreportergenerelativeactivitydrivenbycyclinD2promoterfragmentunderTSAtreatmentdiminishedwhenitwastruncatedto-524bp.ConclusionThein

7、creasedmRNAlevelofcyclinD2ispromoteractivitydependent.TheTSAresponseregionofcyclinD2locatedfromthe-720bpto-524bp.［Keywords］cyclinD2；promoter；reportergene;TrichostatinATheresearchwassupportedbyfoundationfromNationalScienceFoundationofChina(31050010)andHebeiEducationDepartm

8、ent(2010154)andNorthChinaMedicalCollege(BS06002).Correspondingauthor:GuoZhiyi,Tel:86-315-3725747