tnf-α促进乳腺癌细胞的干性及其分子机制的研究word论文.docxVIP

TNF-α促进乳腺癌细胞的干性及其分子机制的研究硕士研究生：张晨雨导师：吴亚群教授华中科技大学同济医学院附属同济医院甲状腺乳腺外科中文摘要目的：通过外源性TNF-α 刺激乳腺癌细胞，研究其促进乳腺癌上皮细胞自我更新的作用及其分子机制。方法：用人分泌型细胞因子TNF-α 刺激乳腺癌细胞后，行成球试验（sphere formationassay），并检测其CD44+CD24-的细胞群（即肿瘤干细胞群）的比例，比较处理前后干细胞表型及成球能力变化；通过Western blot和Real-Time PCR检测TNF-α 处理后pAKT和EZH2 表达水平；通过比较干扰EZH2表达前后乳腺癌细胞中CD44+CD24-细胞群的比例及成球能力，阐述TNF-α调节乳腺癌细胞自我更新的机制。结果：外源性TNF-α 刺激T47D 细胞和MCF-7 细胞后，乳腺癌细胞球体的形成能力明显增加，CD44+CD24-乳腺癌干细胞群的比例亦显著提高；外源性TNF-α通过激活AKT 信号通路来上调EZH2的表达、siRNA介导的EZH2 表达下调能够阻断TNF-α 对乳腺癌细胞自我更新能力的促进。结论：TNF-α通过活化AKT信号通路，上调EZH2表达，增强乳腺癌细胞自我更新能力。关键词：TNF-α；乳腺癌干细胞；丝氨酸/苏氨酸蛋白激酶；EZH2Exogenous stimulation ofTumornecrosis factor-α promotes breast cancerstemcellsCandidate:Zhang ChenyuSupervisor:Prof.WuYaqunThyroid andbreastsurgery of Tongji hospital, Tongji MedicalColledge, Huazhong University of Science and TechnologyAbstractObject:ExogenousstimulationofTNF-αfacilitatetheacquisitionofself-renewal potentialinbreastcancercells,providingusefulinformationforilluminatingthe mechanism ofit.Methods:BreastcancercellsMCF-7andT47Dwerestimulatedbyhumansecreted Tumornecrosisfactoralpha(s-TNFα),afterthenthesphereformationassayoftumorcells andtheproportionofCD44+CD24- phenotypecellsdetectedbyflowcytometrywhichwith thecharacteristicsofbreastcancerstemcellwereusedtoevaluatethecancerstemcells formationabilityoftheposts-TNFαstimulatedbreastcancercells.Quantitativereal-time PCRandwesternblotwereusedtochecktheexpressionofpAKTandEZH2afters-TNFα;WeknockdownEZH2bysiRNAinbreastcancercells，thenobservedthechangeofbreastcancer stem cells upon TNF-α stimulation, to explore the molecular mechanisms of exogenousTNF-α stimulation promotes breastcancerstemcells.Results:AfterexogenousTNF-αstimulatingT47DandMCF-7cells,theformation abilityofmammospherewasobviouslyincreased,andtheCD44+CD24-breastcancerstem cellpopulationwasalsosignificantlyincreasedcomparedwiththeblankcontrolcells. ExogenousTNF-αincreasedtheexpressionofEZH2byactivatingAKTsignalingpathway. Inaddition,usingsiRNAtointerferencetheexpressionofEZH2canblocktheability