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华中科技大学博士学位论文卵巢癌化疗耐药相关分子机制的探讨姓名：周婷申请学位级别：博士专业：妇产科学指导教师：王世宣2010-05_____“√”1“”25%70301.2.3.4.BA/2“”Bub1Bub1B2BB2Bub1A/BB2Bub1A2780Bub1A/BA2780A2780/DDPA2780/TaxolC13KOV20083Bub11906Bub1-3Mad1-3Bub12Bub1siRNApEGFP-Bub1-shRNABub1pEGFP-Bub1-shRNAG418MTTHochest33342A2780SKOV34G2/MBub1A2780SKOV3G2/MA2780SKOV3G2/MpEGFP-Bub1-shRNAG2/MBub1Part1:TheeffecsofBub1onPaclitaxel-resistanceinhumanovariancancercellsAbstractBackgroundandObjectiveThespindlecheckpoint,knownasasurveillancemechanism,iscrucialforensuringthechromosomesegregationprocess.Paststudiesindicatedthatthespindlecheckpointcontrolsthecellcycle.Thewell-characterizedcomponentsofthespindlecheckpointincludeBub1,Bub2,Bub3,Mps1,Mad1,Mad2andMad3.ThefamilyofMad(mitoticarrestdeficient)andBub(buddinguninhibitedbybenzimidazole)proteinswereidentifiedbyyeastmutagenesisscreensformutantsunabletosurviveatemporaryexposuretomicrotubuletoxin.Bub1isthepaltformofspindlecheckpoint.Therearemanycontroversialissuesonthespindlecheckpoint.Theinnnermechanismsareyetwaitingtobeexplored.Paclitaxelsucceededinthetreatmentofsolidtumors,includingbreastcancer,ovariancancerandsoon.Paclitaxelisamicrotubestablereagent,whichblokingcellcycle5tokillthetumor.Somestudiesdemonsteatedthatthekillingeffectsdependonafunctionalspindlecheckpoint.Themutantisrare,buttheofspindlecheckpointoftenoccour.TherearenoreportsontheBub1-mediatedchemoresistanceathome.BothoftheefficiencyandtimelengthofsiRNAisnotenough.ThepEGFPBub1-shRNAplasmidweusedcanobviouslyenhancethesilentefficiency,supplypowerfultooltoprobethemechanismsofpaclitaxel-resistanceinovariancancercells.Methods1.TheimmunohistochemistrystainingofBub1;2.Theconstructionoffull-lengthandpEGFP-Bub1-shRNAplasmids;3.Applypaclitaxelwithdifferentconcentrationgradienttoaffecttumourcells,choosetheconcentrationwhentherateofapoptosisandG2/Mwashighestandwithoutobviousapoptosis;4.ApplyMTTandFACSmethodtotesttheapoptosisandcellcycleoftumourcells;5.ApplyHoechst33342stainingmethodtotestthecellmitosisandapoptosis.ResultsAfterthetreatmentofcertainpaclitaxel,therateofproliferationinhibitionandapoptosisincreased.Thevolumeofapoptosiscellshrunk,thelinksdissolvedthatisolatefromcellsaround,andfollowingthat,thedensityofcytoplasmincreasedandthenucleoplasmtocondense.ThecellcycleofapoptoticcellsmainlyinG2/Mphase.Thedrug-sensitivityofA2780andSKOV3reducedaftertransfectedwithpEGFP-Bub1-shRNAplasmid.ConclusionBothA2780andSKOV3cellscouldbeinducedintoapoptosisbyacertaindosepaclitaxel.ThecellapoptosisandarrestinducedbypaclitaxelcouldbereversedbytransfectingwithpEGFP-Bub1-shRNAplasmid.6Keywordspaclitaxel;chemoresistance;Bub1;ovariancancer1.DH5αpEGFP-C1InvitrogenTakaraG418TRIzolTMGIBCOMTTDMSOPISigmaPCRBub1Chemiconbeta-actinSantaCruz2005-20085010%4µmHE202010Bub11500EDTAPBS2.CO2HeraeusMettlerToledoOlypusHyperion4800PCRBiometraBiometraBiometra0.22µM7NeicaNeicaOlympus-80HarrsPharmaciaBiotechUVPGDS-8000BDFALCONOlypusXDS-1SigmaLabconco3.(1)PBSph7.2―7.4NaC137mmol/LKCl2.7mmol/L,Na2HPO44.3mmol/L,KH2PO41.4mmol/L.(2)0.01mol/LCB,PH6.0,1000ml3g,0.4g(3)0.5mol/LEDTAPH8.0700ml186.1gEDTA,10mmol/LNaOHph9.0,1000ml.(4)1mol/LTBSph8.0800ml121gTris1NHClPH8.0,1000ml(5)3%―H2O230%H2O280%(1)60min6020min---10min,10min---5min---95%min---75%5min(2)PBS25min(3)PBS3%H2O25-10min38(4)0.01PH6.09510-15min(5)PBS5min(6)20min(7)Ι50µl,14371(8)PBS32min(9)3720min(10)PBS32min(11)SABC20-3020min(12)PBS45min(13)DAB(14)4.pEGFP-Bub1-shRNAGenebankBUB1mRNABUB1shRNABub1-shRNA1GAGTGATCACGATTTCTAA2CCAUGGGAUUGGAACCCUGTT3CCAGGCUGAACCCAGAGAGTTshRNARNAi-ReadypSI-REN-DNR-DsRed-ExpressVector,PCRU6shRNAECORMluECORMlupEGFP-C1T43hDH5α0CaCl2DNADNase42DNA250mlLB250mlAmpkana100mg/ml,---3---9100µl50ngDNA3DNADNA30min---EP4290---EP12min---400mlLB3745min---AmpkanaLB---371216------1690952EP250µl34µgDNA6µl5min420min6546G418247080G41810A2780SKOV3G418400600µg/ml11102G418500µg/ml347G4181041014G418196G418PBSRing12minG418962RT-PCRWesternblotP21mRNA32×105EP1500800rpm/min5minPBS100µl3BSA/PBSFITC30min800rpm/min5minPBS500µlPBS5.A2780EuropeanCollectionofcellCulturesECACC10%RPMI1640375%CO290%A2780A278062×105/)70%Lipo2000PEGFP-C1PEGFP-Bub1-shRNAA2780500mg/LG41814pEGFP-C1/A2780pEGFP-Bub1-shRNA/A2780A2780SKOV311ATCC,10%FBSMaCoy'5a,375%CO2,3×105/65060%8µL4.0µg48h500mg/LG418200mg/L26.Hoechst3334212422.5*105/375CO22431µM2436484PBS415min5PBS6Hoechst333423710min7PBS5min*350Neica7.MTT8*103/96375%CO224hPBS2448MTT310mg/mlMTT10µl1mg/ml375%CO24-6DMSO150µl30min570nmOD,=(/)×100%,8.FACS12(flowcytomet)()()1500rpm/min5minPBS175%1ml-201500rpm/min5minPBSRNaseA0.05g/L10µlPI250µl25µg/mlPBS240µl30min43BD3a)b)annexinVc)PI9.RT-PCR48h,TrizolRNAM-MLAPCRGAPDHBub1Bub1:5'-TAAACCCACAGGAGCCAGGAC-3'5'-CAAGCCTCAACGCCCAACT-3':955min9530s56.545s,7230s,297210minGAPDH::5'-ACGGATTTGGTCGTATTGGG-3':5'-TGATTTTGGAGGGATCTCGC-3':955min9530s5445s,7230s307210minPCR1.5%3RNA131800rpm/min5min2PBS800rpm/min5min31mlTrizol5min40.2mlCHCL35min5412,000rpm/min15min6EP70.5ml2010min8412,000rpm/min15min975DEPC1ml10412,000rpm/min10min1112DEPC3050µlRNA13-80RT-PCR12PCR(10pmol/L)1µl(10pmol/L)1µl10×PCRbuffer5µl10mmol/LdNTP1µlMgCl23µlcDNA1µlTaq1.5µ