Cyclin E2和Survivin在急性白血病的表达及其相关性.docx
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Cyclin E2和Survivin在急性白血病的表达及其相关性.docx
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CyclinE2和Survivin在急性白血病的表达及其相关性
CyclinE2和Survivin在急性白血病的表达及其相关性
【摘要】为了解细胞周期蛋白E2和存活蛋白在急性白血病中基因表达及两者间的相关性,采用逆转录-聚合酶链反应的方法检测了84例成人急性白血病患者和20例正常人的cyclinE2和survivinmRNA的表达。
结果表明:
①cyclinE2在急性白血病中表达的阳性率高于对照组;survivin在急性白血病中表达的阳性率高于对照组;②在急性白血病中cyclinE2的表达与survivin的表达呈正相关性,r=;③cyclinE2阳性组缓解率低于cyclinE2阴性组,cyclinE2阳性组复发率高于阴性组;复发组cyclinE2表达率在三组中最高,持续缓解组cyclinE2表达率在三组中最低,④cyclinE2在急性髓系白血病患者表达率低于急性淋巴细胞白血病患者的表达率;与发病时白细胞计数未见有统计学意义的相关。
结论:
首次证实cyclinE2在急性白血病中有异常表达,且对临床预后有不良影响;cyclinE2在急性白血病中的表达与survivin有正相关,提示cyclinE2在急性白血病中有可能作为一种微小残留病变的检测指标。
【关键词】存活蛋白
Asamalignanthemophaty,acuteleukemia(AL),especially,acutelymphocyticleukemia underwentahighrelapserateandashortdisease-freesurvival(DFS),inspiteoftheimprovementinclinicaloutcomebenefitedfromthedevelopmentofhematopoieticstemcelltransplantation(HSCT)andchemical relapseandprolongingDFSstillisthefocusofAL residualdisease(MRD)becameaneffectiveindicationforthefoundationofpost-remissiontherapyregulationsincetheroutinemorphologicalexaminationoftheperipheralbloodandbonemarrowsamplefromtheremissionpatientwas thefusiongenesuchasAML1/MTG8andPML/RARαasMRDmarker,wasonlyusedinM2,M3,soitisvaluabletosearchanothernewMRDmarkerfor theotherhand,RNAinterference(RNAi)asanewandprospectivegenetherapyforcancerhasbecomeanewfocusofthestudyofALtherapy[1].Searchingforaneffectivenewtargetforatumor-specificRNAiisworthdevelopingforleukemia,asatypeofG1cyclin,cyclinE2wasfoundouttobethepotentialmarkeroftumorssinceitpresentedapositiveexpressioninthecelllinesofsolidtumorsandanegativeexpressioninproliferatingnormalcells[2].However,therehasbeennoreportonitsexpressionandrolein ordertofindouttheroleofcyclinE2intheprogressionandclinicaloutcomeofAlpatients,reversetranscriptionpolymerasechainreaction(RT-PCR)isadoptedtotesttheexpressionofcyclinE2andsurvivinmRNAin84adultpatientswithAL,20normalpersonsascontrolsandleukemiacellline,weinvestigatedtherelationshipbetweentheexpressionofcyclinE2andtheclinicalprogressionofAL studyofcyclinE2canprovideapreliminarytheoreticalbasisforsearchinganewtargetofALgenetherapyandanewMRDmarker.
MaterialsandMethods
Patientsenrolled
84adultinpatientswithALfromApril2002toMarch2003intheSecondAffiliatedHospitalofHebeiMedicalUniversitywereenrolledinthis diagnosesofthesepatients[50malesand34females,14-62yearsold(meanof years)]wereconfirmedbymorphological,cytochemicalandimmunologicmarker totheFrench-American-British(FAB)classification,59patientswerediagnosedasacutemyelocyticleukemia(AML),including2casesofM1,12casesofM2,21casesofM3,11casesofM4,11casesofM5,1casesofM6,1casesof patientswerediagnosedasacutelymphocyticleukemia(ALL)including4casesofL1,19casesofL2,2casesofL3;60patientswithdenovoAL,16relapsecases,8casesofcontinuouslycompleteremission(CCR)(timeofdisease-freesurvival≥3years).20normalpersonsaged15-60years(meanof35yearsold)containing11malesand9femalesservedas:
allthesepatientswithAML,exceptM3,receivedstandardADEregimen(cytarabinearabinoside150mg/m2for7days,daunorubicin40mg/m2for3daysandetoposide75mg/m2for3days-7days).PatientswithM3receivedall-trans-retinoicacid(20-40mg/day)andarsenictrioxide(10mg/day)asremissioninduction withALLreceivedVITPregimen(vincristine mg/m2for1dayofeachweek,iphosphamide g/m2for3daysofeachweek,pirarabicin20mg/m2for3daysofeach2weeks,prednisone1mg/kgfor14daysandthengraduallydecrementuntiltheendofregimenthatlastsforabout28days).
Cellsandcellculture
Bonemarrowsamplesfrom84patientswithALand20controlswereusedformononuclearcells(MNC) mlofthisfreshheparinizedbonemarrowsampleswereseparatedbyFicoll-Hypaque(PharmaciaBiotech,Uppsala,Sweden)andwashedtwicewithphosphate-bufferedsaline(PBS),followedbyextractionofwholecelllysates,andtheMNCconcentrationwas106cells/cellsamountedtoover80%inbonemarrowsamplesfrom84ALpatientsassesedbyWright′sstaining,andthentheMNCswerefrozenaftercentrifugationandstoredat-80℃untilfurther celllinewasobtainedfromtheInstituteofHematology,ChineseAcademyofMedical weremaintainedat37℃inahumidifiedatmospherecontaining5% growingsuspensionculturesofleukemiacellswerepropagatedbyreseedingat5×105cells/mlevery3-5days,inRPMI1640mediumsupplementedwith10%fetalbovineserum,50U/mlpenicillinG,and50μg/mlstreptomycin cellswereharvestedatlogarithmicgrowthphase,separatedbyFicoll-HypaqueandwashedtwicewithPBS,thenfrozenaftercentrifugationandstoredat-80℃untiluse.
RNAextraction
TotalRNAwasextractedfromMNCandK562cellbyTRIZOLreagent(Gibco),accordingtotherecommendationsofthemanufacturer.TheconcentrationandpurityoftotalRNAwasdeterminedbyUVspectrophotometry,TheratioofA260/A280reached Theelectrophoresispatternon%agarosegelstainedbyethidiumbromidewasusedfordeterminingtheintegrityoftotalRNAshowingtwobandsof18Sand28Sinthe RNAwasfrozenat-80℃untilfurtheruse.
Semi-quantitativeRT-PCR
Reversetranscriptionreaction20μlreversetranscriptionreactionsystemincludingtotalRNA1μg,M-MLV200U,dNTPs2mmol/L4μl,RNasin(Promega)20U,randomprimer μg,5×RTbuffer4μlwaswarmedforreactionat37℃,60min,thenstopat95℃for5 productswasfrozenat-20℃untilfurtheruse.
Polymerasechainreaction25μlPCRsystemincludingreversetranscriptionproducts2μl,oligonucleotideprimer25pmol,TaqDNApolymerase(Huamei)1U,dNTPs mmol/L,10×PCRbuffer μl(MgCl215mmol/L,pH,Tris-HCl100mmol/L,KCl500mmol/L,BAS20μg/ml),reactionconditionasfollows:
94℃40sec,55℃40sec,72℃60sec,totally29cyclesforcyclinE2andβ-actin;94℃30sec,55℃30sec,72℃60sec,totally35cycles,72℃10minforsurvivintostopthe eachsetofPCRreactions,parallelreactionusingdouble-distilledwaterinsteadofthecDNAtemplatesolutionasanegativecontroltoassurethequalityofthe primersweresynthesized:
senseprimer:
5′-TTGGCAGGTGCCTGTTGAAT-3′,antisenseprimer:
5′-AGCCAGTCCCCCACAGCAT-3′.Theamplifiedproductshouldbe465bp[3].cyclinE2primerwasdesignedwitholigo accordingtogenegi3885975:
senseprimer5′-TGGCTTTTAGAGGTATGTGAA-3′,antisenseprimer5′-TAATGAATCAATGGCTAGAAT-3′product426bp,β-actinsenseprimer5′-TCATCACCATTGGCAATGAG-3′,antisenseprimer5-CACTGTGTTGGCGTACAGGT-3′,product155bp。
PCRanalysisofproducts
Theelectrophoresisof10μlPCRproductswasperformedat85Vfor90minin2%agarosegelcontained mg/Lethidium thebandswereviewedandphotographedunderUVillumination.ThetranscriptswereestimatedtheopticaldensityratioofcyclinE2orsurvivintoβ-ratioofpositivestandardwaslargerthan
Statisticalanalysis
Thedatawasregisteredasmean±betweendifferentgroupswereevaluatedbythechi-squaretest,correctionforcontinuitychi-squaretest,andexactprobabilitiesin2×2 test:
analysisofPearsoncorrelation,Pvaluesoflessthan wasconsiderdstatistically analysiswasperformedusingSPSS computersoftware.
Results
ExpressionofcyclinE2andsurvivinmRNAinK562cells
TheexpressionofbothcyclinE2andsurvivinintheK562celllinewaspositive,andcouldbeusedasapositivecontrol,showedinFigure1,2.
ExpressionofcyclinE2inALandthecontrol
ExpressionofcyclinE2mRNAwasdeterminedin84casesofALwere20negative(Figure1).TherateofexpressionofcyclinE2inALwas%andmuchhigherthan0%incontrols(χ2=,).
ExpressionofsurvivininALandthecontrol
ExpressionofsurvivinmRNAwasdeterminedin84casesofAL,outofwhich61caseswerepositiveand23caseswerenegative,that6in20controlswerepositiveand14werenegative(Figure2).TherateofexpressionofsurvivininALwas%andmuchhigherthanthatincontrol30%,.
ThecorrelationbetweenexpressionsofcyclinE2andsurvivininAL
ExpressionofcyclinE2wassignificantlypositivelycorrelatedwithexpressionofsurvivinin84casesofAL(Figure3)(r=,).
%(24/43)ofremissionof43cyclinE2-positivepatientswithdenovoALwerelowerthan%(15/17)of17cyclinE2-negativepatientswithdenovoAL;thedifferencewassignificant(χ2=,).Allof14patientswithdenovoM3obtainedcompelteremissionafterreceivinginduction rateofremissionof8cyclinE2-positivepatientswithdenovoM3was100%(8/8),andthatof6cyclinE2-negativepatientswithdenovoM3wasalso100%(6/6),andtherewerenodifferencebetweenthem.Theremissionrateof35cyclinE2-positivepatientswithdenovoALexceptM3was%(16/35)lowerthan%(9/11)of11cyclinE2-negativepatientswithdenovoALexceptM3;andthedifferencewassignificant(χ2=,).39ofthe60patientswhoobtainedremissionhadbeenclinicallyobservedfor24months,anditprovedthat24remissionpatientswithdenovoALincyclinE2-positivegrouphasarelapserateof%(10/24),whichishigherthan20%(3/15)ofthe15remissionpatientswithdenovoALincyclinE2-negativegroup,butthedifferencewasnotstatisticallysignificant(exactprobabilitiesin2×2table,).16remissionpatientswithdenovoALexceptM3incyclinE2-positivegrouphasarelapserateof%(10/16),whichishigherthan%(3/9)ofthe9remissionpatientswithdenovoALexceptM3incyclinE2-negativegroup,ExpressionofcyclinE2inrelapsegroup,denovoALgroupandCCRgroupwasshowedin rateofcyclinE2expressioninrelapsegroupwasthehighestinthethreegroups,(100%vs%,χ2=,;100%vs0%,exactprobabilitiesin2×2table,);ther
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- Cyclin E2和Survivin在急性白血病的表达及其相关性 E2 Survivin 急性 白血病 表达 及其 相关性