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ID:33406397
大小:23.27 MB
页数:79页
时间:2019-02-25
《肿瘤细胞靶向性锰配合物诱导细胞死亡与抑制糖酵解的机制》由会员上传分享,免费在线阅读,更多相关内容在行业资料-天天文库。
1、江苏大学硕士学位论文ABSTRACTobject:TostudytheanticanceractivityofthenovelmanganesecompoundAdpaMnAc([(Adpa)2Mn2(Ac)(H20)2](Ac))anditspossiblemechanisms;ThepossiblemechanismsoftheHypoxia-induciblefactors(HIF)prolylhydroxylase(PHD)-mimeticmanganesecompoundAdpaMnCl([(Adpa
2、)Mn(C1)(H20)])wereinvestigated,includingtheeffectsontheglycolysisandmitochondria.Methods:FourhumancancercelllinesincludingU251,HeLa,HepG2andA549cellsweretreatedbyAdpaMnAc.CancercellsproliferationsweredetectedbyMTTassayandthe50%inhibitoryconcentrations(IC50)w
3、erecalculated.ComparetheinhibitionrateofAdpaMnAconnormallivercellsWRL一68andlivercancercellsHepG2.WesternblottodetecttheexpressionoftransferrinrecepotorI(TfRl)andDivalentmetaliontransporters-1(DMT-1);Deferoxamine(DFO)orferriccitratewereusedtoupregulateordownr
4、egulatetheTfRIexpressionsandthenanalysedthechangeoftheanticanceractivityofAdpaMnAc.ThemorphologicalandnucleuschangesinH2B-GFP—labledHeLacellswereobservedbyalivecellsystem(LCS).CellapoptosisWasdetectedasfollows:nucleusmorphologicalchangeswereobservedbystainin
5、g研mHoechest33342,theratesofcellapoptosisweredetectedbystaining耐mAnnexinV/PI,releasingofCytochromeC(CytoC)frommitochondriaandexpressionsofprocaspase-9:AutophagiccelldeathWasexaminedbymonodansylcadervarine(MDC)labelingandLysotrackerRedstaining;autophagy—relate
6、dproteinsLC3andBeclin-1evaluationwimwesternblotandGFP—LC3plasmidtransfection.Themitochondriafunctionswereanalysed:Mitochondriamembranepotential(MMP),ATPlevelsand02consumptionweremeasuredbyJC一1staining,LuciferaseassayandClarkoxygenelectroderespectively;Intrac
7、ellularfreecalciumconcentrationbyFluo-3/AM;Reactiveoxygenspecies(ROS)byDCFH-DAstaining;Mitochondrialswellingwereusedtofindoutitsinteractionwitllmitochondfialoutermembrane.Ontheotherhand,weanalyzedtheinteractionbetweenAdpaMnClandhypoxia-induciblefactor-la(HIF
8、—la)bymolecularmodeling,theactivityofHIF—labyDualLuciferaseReporterassay,themRNAlevelbyRT-PCILandtheproteinslevelbywesternblot;Detectedtheexpressionlevelsofglucosetransporterl(GLUTl)andlactate肿瘤
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