TheExpressionofhTERandhTERTmRNAinHumanTumorswithTissueMicroarray.doc

The Expression of hTER and hTERT mRNA in Human Tumors with Tissue MicroarrayFeb.2006,Volume3,No.2(SerialNo.is)JournalofUS—ChinaMedicalScience,ISSN1548—6648,USATheExpressionofhTERandhTERTmRNAinHumanTumorswithTissueMicroarrayJunYang一KangminWangI.BaoshanSu|.~ngHuang|.ShipingZhaoiYiliWan.LvshengSi2Abstract:Toexploreexpression.patternsofhTERandhTERTmRNAinhumantumorsbytissuechipwithinsituhybridization(ISH),simultaneously,evaluatetheapplicabilityandvalueoftissuechipinISH.Tissuechipsconsistedof230specimensofhumantissues(10casesnormaltissues,13casesbenigntumors,207casesmalignanttumors)werepreparedthroughthepatenttissuearrayer.TheexpressionofhTERandhTERTmRNAinspecimensweredeterminedbyISH.TheexpressionofhTERandhTERTmRNAinnormaltissues,benignandmalignanttumorswererelatedtocharacterizationoftumor<O.005).ThedistributionofhTERandhTERTpositivecellswasregulatedfromlimitedinbasallayertodiffusedthroughoutmostlayersofsquamousepitheliumiscorrelatedwithincreasingseverityofhistopatllologicchanges.Thatmeanstheexpression—patternsoftissuechipprovidesarapidandpowerfulhistologicalmethodthatwouldbeusedinISHandplayanimportantroleintumorbiology.And,theexpression—patternsofup-regulationanddysregulationofhTERandhTERTofisanimportanthistologysigncouldbeusedasausefulmolecularmarkerfordiagnosisandidentificationhumantumor.Keywords:tissuechip(tissuemicroarray);tumor;humantelomerase,insituhybridization(ISH)In1998,Kononen【I】reportedanewtypemicroarraytechnologynamedastissuemicroarray(T/viA).alsocalledtissuechip(TC).Inthispaperweusingtissuemicroarrayandinsituhybridization(ISH)technologytodetecttheexpressionofhTERmRNAandhTERTmRNAin230casesofmultitypesofhumantumorandnormaltissue.MATEⅪALSANDMETH0DS1?Specimens230casesofParaffin—embeddedtissuesfromDepartmentofpathologyofSecondhospitalofXi,anJiaoTongUniversitywereinthisexperiment,amongwhich207caseswereadvancedcarcinoma(gastricadenocarcinoma22,oesophagussquamouscarcinoma21,duodenmnadenocarcinoma3,colonadenocarcinomal2.recto.adenocarcinoma5,hepatocarcinoma7,Hepatocholangiocarcinoma1,cholecystadenocarcinoma4,pancreasadenocarcinoma2,larynxsquamouscarcinoma8,maxillarysinussquamauscarcinoma1,amygdalosquamouscarcinoma1,lungsquamouscarcinoma4,lungadenocarcinoma2,transitionalcellcarcinomaofbladderl2.transitionalcellcarcinomaofureter2,transitionalcellcarcinomaofrenalpelvis1,clearcellcarcinomaofkidneyTheExpressionofhTERandhTERTmRNAinHumanTumorswithTissueMicroarray17,adenocarcinomaofmammary26,thyro—papillarycarcinoma14,thyro-folliculicarcinoma3,medullarythyroidcarcinoma3,endometriamcarcinoma2,cervixsquamouscarcinoma5,Vulvasquamouscarcinoma1,ovario—cystadenocarcinoma3,leiomyosarcoma2,Bcelllymphoma5,medulloblastoma5,neuroblastoma2,retinoblastoma1,Wilmstumor2,spermatocytoma1,melanoma2.neurofibrosarcoma1,chondrosarcoma1.3casesofmalignancyfibroushistiocytoma),13casesofmiscellaneousbenigntumorsand10casesofnormaltissue(mainlyfrommucosatissues)ascontro1.2.TissuechippreparationAccordingtothemethodestablishedinourinstitute,weprepared4x6type(rowxcolumn)tissuechip(23samplesforeachchip).Totally,20slideswerepreparedwithfollowingmethod:FirstlytheHEsectionsofthe230casesofparaffinembeddedspecimenswereread,andthecorrespondingfittingregionsoftheparaffinembeddedblockswereexcerpted.Then,thetissuearrayerpreparedbyourselveswasusedspeciallyfordiggingouttissuecylinder1.5mmindiameterand3mminlengthfromthedonatorblocks.Thenevery23cylinderswereembeddedinoneparaffinblockandlabeled.Furthermore.paraffinsection51.tmwaspreparedandmountedontheslidescoatedwithpoly-lysine.3.InsituhybridizationhTERandhTERTISHKitscontaintheeDNAprobeslabeledwithdigoxinwerepurchasedfromtheDepartmentofpathology,Beijingmedicaluniversity,andtheISHwascardedoutjustfollowingthedirectionofmanufacture.Thesectionsweredeparaffined,hydratedwithO.1mmol/LHC1,digestedwithproteinaseK(1001.tg/m1),refixedwith4%formalin.washingthesectionsweredehydratedwithgradedethanolsolution.201.tlofhybridizationsolutionwasaddedoneachsectionandhybridizedovernight4"C,afterblockingwithnormalhorseserum,thesectionswerereactedwithanti-Dig-AP,developedwithNBT/BCIP.Sectionshybridizedwithprehybridizationsolwithoutprobes,andsectionstreatedwithRNAaseandhybridizedwiththeprobesinparallelwereusedasnegativecontro1.4.EvaluationofthequalityoftissuemteroarrayComparethetissuechipsectionshybridizedwithprobeswerecomparedwiththesectionpreparedbyconventionalmethodinthefollowingaspects:wrinkles,defection.sheddingoffandespeciallywhethertheresultsofhybridizationwereconsistentwiththe.realorno.'5.DeterminatetheresultsofISHWhenpurpleproductsappearedinthecytoplasms.itwasjudgedaspositive.Theintensitywasgaugedinto4grades:negative(.):nopurplecolorcouldbedetected;weaklypositive(+):purplegranulesappearedinthecytoplasmoftumorcellsandpositivecellswerelessthan25%oftotalcells;mediatedpositive(++):purpleproductsappearedinthecytoplasmoftumorcellsandpositivecellswerebetween25%to50%oftotalcells;stronglypositi。