过表达daxx对cholmβcd诱导巨噬细胞凋亡影响word论文.docxVIP

过表达Daxx对胆固醇β环糊精诱导巨噬细胞凋亡的影响硕士研究生：崔淑华导师：庹勤慧副教授摘要目的：观察Daxx对Chol:MβCD 诱导RAW264.7 细胞荷脂与凋亡的影响，为延缓动脉粥样硬化的病理改变提供理论依据。方法：人源性Daxx基因重组质粒稳定转染RAW264.7细胞，应用RT-PCR和Western blot检测稳定转染细胞内Daxx的表达。然后用Chol:MβCD与RAW264.7 细胞共同孵育48h，建立RAW264.7 细胞荷脂模型，油红O染色和酶荧光法检测细胞内荷脂蓄积情况。MTT检测细胞活性，流式细胞术观察细胞凋亡。Western blot 测定ASK1，JNK，Caspase3 的表达情况。结果：转染细胞经G418筛选14 天后，RT-PCR 和Western blot检测转染细胞内Daxx明显高表达，表明稳定转染成功。Chol:MβCD 与RAW264.7细胞共同孵育48h后，结果发现Daxx高表达组细胞胆固醇蓄积明显，并且细胞活性下调，凋亡细胞数目增加；同时Daxx转染细胞组ASK1，JNK，Caspase3蛋白表达明显增高。结论：Daxx可介导Chol:MβCD 诱导的巨噬细胞凋亡，其凋亡通路可能与Daxx 上调ASK1 表达，继之增加下游JNK活性，进一步激活Caspase3有关。关键词：Daxx；巨噬细胞；凋亡；胆固醇2EffectofoverexpressedDaxxonapoptosisofRAW264.7cellsinducedbycholesterol:modelofβ-cyclodextrin.ABSTRACTObjective:TheeukaryoticvectorofDaxxwasconstructedtobestablytransfected intoRAW264.7cells.Theaimofthepresentstudywastodeterminetheeffectof Daxxon apoptosis of RAW264.7 cells induced byChol:MβCD .Methods:TheeukaryoticvectorofDaxxwasconstructedtobestablytransfected intoRAW264.7cells.WedetectedtheexpressionofDaxxincellstransfectedwith reorganizedvectorusingRT-PCRandWesternblottingtodecidewhetherthe eukaryoticvectorissuccessfullyconstructed.Then,therecombinanttransfected RAW264.7cellsweretreatedwith20mg/LChol:MβCDfor48h.OilRedODyewas usedtoshowthelipiddropletsinChol:MβCDtreatedcells.Enzymefluorescent analysiswasperformedtodeterminethecontentofcellulartotalcholesterolandfree cholesterol.RAW264.7 macrophagesgrowth inhibition was measured byMTTassay. TheapoptosiseffectofRAW264.7murinemacrophagesinducedbyChol:MβCDwas analyzedbyflowcytometricanalysis.TheexpressionofASK1,JNKandCaspase3 were assayed byWesternblotting.Results:RT-PCRandWesternblottinganalysesshowedthattheexpressionofDaxx wassignificantlyincreasedingene-transfectedRAW264.7cells.Toexploretheeffect ofChol:MβCDoncellulargrowthinRAW264.7cells,thecellsweretreatedwith 20mg/LChol:MβCDfor48h.OilRedOshowedthatthelipiddropletsweremorein transfectedRAW264.7cellsthanincontrolandvectorgroup.Intracellulartotal cholesterol(TC)andfreecholesterol(FC)signi