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1、The Expression of hTER and hTERT mRNA in Human Tumors with Tissue MicroarrayFeb.2006,Volume3,No.2(SerialNo.is)JournalofUSChinaMedicalScience,ISSN15486648,USATheExpressionofhTERandhTERTmRNAinHumanTumorswithTissueMicroarrayJunYang一KangminWangI.BaoshanSu|.ngHuang|.ShipingZhaoiYiliWan.LvshengSi2Abstract
2、:Toexploreexpression.patternsofhTERandhTERTmRNAinhumantumorsbytissuechipwithinsituhybridization(ISH),simultaneously,evaluatetheapplicabilityandvalueoftissuechipinISH.Tissuechipsconsistedof230specimensofhumantissues(10casesnormaltissues,13casesbenigntumors,207casesmalignanttumors)werepreparedthrought
3、hepatenttissuearrayer.TheexpressionofhTERandhTERTmRNAinspecimensweredeterminedbyISH.TheexpressionofhTERandhTERTmRNAinnormaltissues,benignandmalignanttumorswererelatedtocharacterizationoftumor<O.005).ThedistributionofhTERandhTERTpositivecellswasregulatedfromlimitedinbasallayertodiffusedthroughoutm
4、ostlayersofsquamousepitheliumiscorrelatedwithincreasingseverityofhistopatllologicchanges.ThatmeanstheexpressionpatternsoftissuechipprovidesarapidandpowerfulhistologicalmethodthatwouldbeusedinISHandplayanimportantroleintumorbiology.And,theexpressionpatternsofup-regulationanddysregulationofhTERandhTER
5、Tofisanimportanthistologysigncouldbeusedasausefulmolecularmarkerfordiagnosisandidentificationhumantumor.Keywords:tissuechip(tissuemicroarray);tumor;humantelomerase,insituhybridization(ISH)In1998,Kononen【I】reportedanewtypemicroarraytechnologynamedastissuemicroarray(T/viA).alsocalledtissuechip(TC).Int
6、hispaperweusingtissuemicroarrayandinsituhybridization(ISH)technologytodetecttheexpressionofhTERmRNAandhTERTmRNAin230casesofmultitypesofhumantumorandnormaltissue.MATEALSANDMETH0DS1?Specimens230casesofParaffinembeddedtissuesfromDepartmentofpathologyofSecondhospitalofXi,anJiaoTongUniversitywereinthisex
7、periment,amongwhich207caseswereadvancedcarcinoma(gastricadenocarcinoma22,oesophagussquamouscarcinoma21,duodenmnadenocarcinoma3,colonadenocarcinomal2.recto.adenocarcinoma5,hepatocarcinoma7,Hepatocholangiocarcinoma1,cholecystadenocarcinoma4,pancreasadenocarcinoma2,larynxsquamouscarcinoma8,maxillarysin
8、ussquamauscarcinoma1,amygdalosquamouscarcinoma1,lungsquamouscarcinoma4,lungadenocarcinoma2,transitionalcellcarcinomaofbladderl2.transitionalcellcarcinomaofureter2,transitionalcellcarcinomaofrenalpelvis1,clearcellcarcinomaofkidneyTheExpressionofhTERandhTERTmRNAinHumanTumorswithTissueMicroarray17,aden
9、ocarcinomaofmammary26,thyropapillarycarcinoma14,thyro-folliculicarcinoma3,medullarythyroidcarcinoma3,endometriamcarcinoma2,cervixsquamouscarcinoma5,Vulvasquamouscarcinoma1,ovariocystadenocarcinoma3,leiomyosarcoma2,Bcelllymphoma5,medulloblastoma5,neuroblastoma2,retinoblastoma1,Wilmstumor2,spermatocyt
10、oma1,melanoma2.neurofibrosarcoma1,chondrosarcoma1.3casesofmalignancyfibroushistiocytoma),13casesofmiscellaneousbenigntumorsand10casesofnormaltissue(mainlyfrommucosatissues)ascontro1.2.TissuechippreparationAccordingtothemethodestablishedinourinstitute,weprepared4x6type(rowxcolumn)tissuechip(23samples
11、foreachchip).Totally,20slideswerepreparedwithfollowingmethod:FirstlytheHEsectionsofthe230casesofparaffinembeddedspecimenswereread,andthecorrespondingfittingregionsoftheparaffinembeddedblockswereexcerpted.Then,thetissuearrayerpreparedbyourselveswasusedspeciallyfordiggingouttissuecylinder1.5mmindiamet
12、erand3mminlengthfromthedonatorblocks.Thenevery23cylinderswereembeddedinoneparaffinblockandlabeled.Furthermore.paraffinsection51.tmwaspreparedandmountedontheslidescoatedwithpoly-lysine.3.InsituhybridizationhTERandhTERTISHKitscontaintheeDNAprobeslabeledwithdigoxinwerepurchasedfromtheDepartmentofpathol
13、ogy,Beijingmedicaluniversity,andtheISHwascardedoutjustfollowingthedirectionofmanufacture.Thesectionsweredeparaffined,hydratedwithO.1mmol/LHC1,digestedwithproteinaseK(1001.tg/m1),refixedwith4%formalin.washingthesectionsweredehydratedwithgradedethanolsolution.201.tlofhybridizationsolutionwasaddedoneac
14、hsectionandhybridizedovernight4C,afterblockingwithnormalhorseserum,thesectionswerereactedwithanti-Dig-AP,developedwithNBT/BCIP.Sectionshybridizedwithprehybridizationsolwithoutprobes,andsectionstreatedwithRNAaseandhybridizedwiththeprobesinparallelwereusedasnegativecontro1.4.Evaluationofthequalityofti
15、ssuemteroarrayComparethetissuechipsectionshybridizedwithprobeswerecomparedwiththesectionpreparedbyconventionalmethodinthefollowingaspects:wrinkles,defection.sheddingoffandespeciallywhethertheresultsofhybridizationwereconsistentwiththe.realorno.5.DeterminatetheresultsofISHWhenpurpleproductsappearedin
16、thecytoplasms.itwasjudgedaspositive.Theintensitywasgaugedinto4grades:negative(.):nopurplecolorcouldbedetected;weaklypositive(+):purplegranulesappearedinthecytoplasmoftumorcellsandpositivecellswerelessthan25%oftotalcells;mediatedpositive(+):purpleproductsappearedinthecytoplasmoftumorcellsandpositivec
17、ellswerebetween25%to50%oftotalcells;stronglypositive(+):darkpurpleproductsexistedinthecytoplasmoftumorcellsandpositivecellsweremorethan5O%oftotalcells.6.StatisticalanalysisDifferencesbetweenthegroupsweretestedbytest.RESUITS1.TheresultsoftissuechlpTissuesample1.5mmindiameterarrayedontheslidesinorder,
18、thetissuestructureisintegrity,18cylinder.T.h.e.E.x.p.r.e.s.s.i.o.n.o.f.h.T.E.R.a.n.d.h.T.E.RTmRNAinHumanTumorswithTissueMicroarraytissuesamplesshedfromslide,therewere221cylindertissuesamplescanbeanalysis,thesheddingrateis3.9%2?4%?thestructureofdonatorparaffinblocksisintegrityandcanbeexperimentedcont
19、inually.Visualizedundermicroscope?thesectionswereveryclearandhadaveryweakbackgroundandawellcontrast(Figure3).Fig?1TheImageofspecimeninthe4x6typeStissuechipsFlg?2hTERmRNAexpressedinoesophagussquamouscarcinoma,darkpurpleproductsexistedInthecytop1asm0ftumorcells.DevelopedwithNBT/BCIP,x400.FIg?3hTERTmRN
20、Aexpressedingastricadenocarclnoma,darkpurpieproductsexistedinthecytoplasmoftum0rceIIs.DevelopedwithNBT/BCIP,x400.2.TheresultsofISHTheexpressionofhTERandhTERTmRNAwasshowedintable1.32Table1TheexpressratioofhTER.andhTERTmRNAin230casesofhumantumorandnOrmaltlssue00000._?_-_.?_-_?_-_-_-V尊1.1.锥l譬蠕磷斡雌蕾!毳.T.
21、h.e.E.x.p.r.e.s.s.i.o.n.o.f.h.TE.Rand.hTERTmRNAinHumanTumorswithTissueMicroarray3.ThehistoriealdistributionofpositivecellsAsshowninFigure2-3,hTERmRNAexpressedincytoplasmoftumorcells,hTERmRNAexpressedweaklyinnormaltissueandlimitedtothebasalcellsmerely.Therearefewweakpositivecellsinbasaleelllayercould
22、befound.Inbenigntumor,hTERmRNAexpressedweakly,thepositivecellswerediffusedlyinmostlayeroftumortissue.Ininvasivecarcinomas,theexpressionofhTERmRNAwasmediatedandstronglypositive,thepositivecellsdiffusedthroughoutmostlayersofsquamousepitheliumasmassshape,nests,hTERTmRNAexpressedincytoplasmoftumorcells.
23、hTERTmRNAexpressionanddistributionhadasimilarregulationofhTERmRNAexpression.hTERTtuRNAexpressedweaklyandlimitedtothebasalcellsinnormaltissuemerely,Inbenigntumor,hTERTmRNAweaklypositivecellswerediffusedinmostlayerintumor.Ininvasivecarcinomas,thehTERrnRNAexpressedmediatedandstrongly,mediatedandstrongl
24、ypositivecellswerediflusedthroughoutmostlayersofsquamousepitheliumasmassshape,nests.SoexpressionofhTRRNAandhTERTmRNAwascolocalizednotonlyincarcinomaregionsbutalsoinbenigntumorandnormalepidermallayersinhistologica1.ThatistosaytherewasaspecialhistoricaldistributionofhTERandhTERTpositivecellsinnormalor
25、tumortissuesDISCUSSION1?TissuemicroarrayTMAwasdesignedfordetectingsamemarkerindifferentspecimensinsitu.UsingTMAtechnology,wecanstudyseveraldozensofspecimensinDNA,RNAorproteinlevelinsitu.Atpresent,thistechniquehasbeenusedinmanyaspects2?.TMAoffersanefficientwaytoexaminealargenumberoftumorspecimensonas
26、ingleslide.However,TMAisbiopsyofabiopsy,sotheimportantthingistoassessthepossibilityandvalidityofTMAespeciallyexamineagroupofheterogeneitytumorwithTMA.Manyauthorsthoughtthatarrangednumerous0.6一mmindiametertissuecorebiopsyspecimensintosingleparaffinblockanavalidatedthatTMAcanbeusedtodetectheterogeneou
27、sorhomogeneousproteinorgeneexpression5-71Furthermore,Kim【8】constructedTMAbyarrangingthecylindricalbiopsiesof2.0mmindiameterintoatissuearrayblock.Inthispaper.weutilizedtheprivatePatenttissuearrayerbemanufacturedbyourselves(PatentNO:L00207528.8,NO:ZL00262393.5),topreparetissuemicroarraysbyarrangingthe
28、cylindricalbiopsiesof1.5mmindiameterintoatissuearrayblocktoensurethespecificityandthehighthroughcharacterofTMA.Inthisstudytherewere460samplesmountedonto20slidesof4x6typetissuechippreparedintota1.18cylindricaltissuesamplesshedoff;thesheddingratewas3.9%2.4%.1/23timesofmaterialsreagentandtimef0rIhecomm
29、onmethodwasusedintheexaminationonly.Sousingtissuechipwecaneconomizetheexperimentmaterial,increasedtheefficienciesoftestmarkedly.ThisresultsuggestedthatTMAaffordedanefficientandexpeditiousmethodforcancerresearch.ThecorrespondingfittingregionsselectedfromthedonatorblokeswereaccordingtotheirHEsections,
30、sothat,TMAtechniquecanavoidthehemorrhageandnecrosisareaincylindricalbiopsytissues,reducetheirinfluenceontheresultsofexperiment.Inaddition,usingthistechnologic,theexperimentcanbedoneundersimilarconditionbyarrayingmanysamplesononeslide,sotheerrorofexperimentcouldbedecreasedtotheutmostextent.PackeisenL
31、gJthoughtthatTMAcanbeusedinsituresearchasatoolforintemalqualitycontro1.Inthispaper,TMAwasusedinthefieldofcancerresearchwithISH,andimprovedtheefficiencyof33TheExpressionofhTERandhTERTmRNAinHumanTumo.r.s.w.i.thTiss.u.e.M.i.c.r.o.a.r.rahistopathologyresearchmarkedly.TheresultssuggestthatISH-TMAisaneffi
32、cientandexpeditioustechniquecanbereliablyusedtoevaluatetheprognosticvalueofexpressedbiomarkers,throughexamininguptoseveralhundredsdifferentclinicalspecimensinasingleslide.Atthepresent,asanefficientandexpeditiousbiochip,TMAhasrecentlybecomeawidelyacceptedstandardtechnology,andcanbeusedforalldifferent
33、typesofinsitutissueanalysis.2.HumantumorandtelomeraseTelomerase,aribonucleoprotein,iscomposedofaRNAcomponent(hTER)andtwoproteinsubunits.Oneofthesesubunits,thecatalyticsubunit(humantelomerasereversetranscriptase,hTERT),representsareversetranscriptase.TheexpressionofhTERTiscloselycorrelatedwithtelomer
34、aseactivity.KammoritmJsuggestthatISH.basedanalysisofhTERTgeneexpressionissuperiortobethTRAPtelomeraseactivityandhTERTmRNART-PCRanalysisasameansofdeterminingtelomerasestatusduringcarcinogenesis.ThusevaluatinghTERTgeneexpressionlevelscouldbeusedasamarkerofmalignantprogressionllJ.Inthispaper,wedetected
35、theexpressionofhTERandhTERTmRNAin207casesofhumanmalignanttumor,13casesofbenigntBmorandl0casesofnormaltissuewithTMAandISH.TheresultsshowedthattheexpressionofhTERmRNAandhTEI玎mRNAup-regulation<0.005)followingtheprogressionoftumor.ThissuggestthattheexpressionofhTERmRNAandhTERTmRNAhasasignificantcorre
36、lationwithoccurrenceandprogressionoftumor.inthispaper,wefoundthattheexpressionofhTERmRNAwasnegativeinl1.4%(23/207)malignanttumors,theexpressionofhTERTmRNAwasnegativeinl7.9%(36/207)malignanttumors,buttheexpressionofhTERmRNAwasweakpositivein55.6%(5/9)normaltissues,theexpressionofhTERTmRNAwasweakpositi
37、vein22.2%(2/9)normaltissues,ThatwastosayhTRandhTERTmRNAwerepresentnotonlyincarcinomatissuesbutalsoinnormalepidermallayers.ManystudieshavealsoshownthathTERandhTERTmRNAcanbedetectedinnon.neoplastictissuesandnormaltissues【,Belair14andNakanoii5thoughtthattheexpressionofhTERandhTERTmRNAhadarelationshipwi
38、ththehighproliferationactivityofcel1.Sowesuspectedthattherepossibleweresomeatypicalhyperplasiacellsorhi.ghhyperplasiacells,fortheatypicalhyperplasiacellsposseshighproliferationactivityastumorcell,thereforetheexpressionofhTERandhTERTmRNAwereupregulationinnormaltissue.Ontheotheraspect,theresultssugges
39、tedthattheup-regulationexpressionofhTERmRNAandhTERTmRNAmerelyplayanbasicroleintheprocessoftumor,andthelevelsofexpressionofthesubunitsoftelomeraseandtheirequilibration,transcriptionandthemodificationposttranscriptionandotherreasonswehadnotknownatpresentmayplayanimportantroleintheregulationoftelomeras
40、eactivityandtheoccurrenceandprogressionofcancerThisresultalsoshownthattheexpressionofhTERandhTERTmRNAwasweakandthepositivecellswerelimitedtothebasalcellsinnormaltissueandbenigntumors.butthattheexpressionofhTERandhTERTmRNAweremediatedandstronglyandpositivecellswerediffusedthroughoutmostlayersofsquamo
41、usepitheliumasmassshape,nestsinmalignanttumors.SotheexpressionofhTERandhTERTmRNAwasco.localizednotonlyincarcinomaregionsbutalsoinbenigntumorandnormalepidermallayersinhistological,andtherewasaspeciaIhistoricaIdistributionofhTERandhTERTpositivecellsinnormaIortumortissues.Theexpression-patternsarecorre
42、latedwithincreasingseverityofhistopathologicchanges.Otherauthorssuggesteditso.Sowesuggestedthattheup-regulationanddysregulationofhTERmRNAandhTEIUmRNAhadanimportantsignificancetodiagnosisbenignormalignanttumor,andperhapstheexpression-patternsofhTERandhTERTcouldbedeterminedasausefullnolecularmarkerfor
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