资源描述:
《葛根素对子宫内膜rl952细胞p450芳香化酶表达的调控作用》由会员上传分享,免费在线阅读,更多相关内容在工程资料-天天文库。
1、葛根素对子宫内膜RL952细胞P450芳香化酶表达的调控作用【摘要】探讨葛根素对子宫内膜RL952细胞P450芳香化酶(aromataseP450,P450arom)表达的影响及其对RL952细胞P450arom基因表达的调控作用。方法:将梯度稀释的葛根素在不同时间点加入RL952细胞中,分别采用实时聚合酶链反应(realtimepolymerasechainreaction,RTPCR)检测药物对RL952细胞P450arommRNA表达水平的影响。以人类基因组DNA为模板扩增人P450arom组织特异性启动子2(PⅡ)转录起始位点上游序列。
2、PCR产物定向克隆到报告基因载体pGL3Basic中,构建人P450aromPⅡ系列报告基因质粒,并经限制性内切酶消化鉴定和基因测序证实。瞬时转染系列重组质粒至RL952细胞,加入葛根素刺激,分析相关转录因子,利用RTPCR及aterialandmethods2.1MaterialsandcellculturePuerarinSigma(St.Louis,MO).LuciferaseAssaySystemsandpGL3BasicVectorPromega(Madison,D).AllchemicalsandenzymesSigma(St.Louis
3、,MO).Phenolredfree(PRF)DMEM/F12,andcharcoalstrippedfetalbovineserum(CSFBS)Gibco(GrandIsland,NY).RL952(ATCCCRL1671TM,USA)humanendometriumcarcinomacellsentedidifiedatmosphereof95%airand5%CO2,at37℃.2.2RNAisolationandquantitativeRTPCRanalysisRL952cellsately70%to80%confluenceprio
4、rtothemencementofeachexperiment.TotalRNA2goftotalRNAprimersfolloanufacturer'sprotocol(MBIFermantas,Vilnius,Lithuania).ThesinglestrandedcDNAePCRtoevaluatetherelativeexpressionlevelsofP450arom(5'GAGAGCATACAGAAGATACACGACTTGand5'GACCAGCCTTCTCTAGTGTTCCA)andhumancjun(5'CCCCCAGCGTAT
5、CTATATGGAAand5'GCTGTCCCTCTCCACTGCAA)paredanβactin(5'CTGGAACGGTGAAGGTGACAand5'AAGGGACTTCCTGTAACAATGCA).RTPCRedaccordingtotheABImanufacturer'sprotocols(AppliedBiosystems,CA).Allsamplesinedintriplicate.PCRspecificamplificationedintheAppliedBiosystems(ABI7300)quantitativeRTPCRm
6、achine.Thereactionsethod[17].2.3SF);1mmol/Lethylenediaminetetraaceticacid(EDTA)].Thisinincubationoniceandcentrifugationat10000×gfor10minat4℃.ProteinconcentrationsinedbyamodifiedBradfordassay(BioRadLaboratories,Inc.,Hercules,CA).Proteinextractsidegel,blottedontoanitrocellulosememb
7、rane(MilliporeCorp,Bedford,USA),andincubated(Biovision,USA)andantiβactin(CellSignalingTechnology,Beverly,USA).ImmunoblotsouseimmunoglobulinG(IgG),folloiluminescence.adison,V(Renillaluciferase;Promega)asaninternalcontrol.Thetransfectionmediumol/Lpuerarinfor12hours.Luciferaseactivi
8、tyeasuredbyDualLuciferase