基于尼龙膜的点杂交条件的建立及优化

1,2(1.,215003;2.,,215007)[]::,,,,:N22(pp2GalNAcT2)K562NB4NIH3T3SGC7901:,,:,[];;[]R318[]A[]167125098(2006)0420509203EstablishmentandOptimizationofDotBlotTechniqueSHAOXue2jun1,WUShi2liang2(1.SoochowUniversityAffiliatedChildren’sHospital,SuzhouJiangsu215003,China;2.DepartmentofBiochemistry&MolecularBiology,MedicalSchool,SoochowUniversity,Suzhou,Jiangsu215007,China)Abstract:ObjectiveToestablishandoptimizethedotblottechnique.MethodsWeusedthepositivelychargednylonmembranestooptimizeexperimentsforthedotblotincludingprehybridizationsolution,hybridizationsolution,washingbuffer,blockingsolutionetal,andthetimesofeverystep.Inordertovalidateitsfeasibility,weanalyzedtheexpressionofppGalNAc2T2indifferenttumorcelllinesbydotblotassayusingtheestablishedhybridizationsystem.Resultswees2tablishedandoptimizedthedotblottechniquewhichhadreliableresultsandlowbackground.ConclusionTheestab2lisheddotblottechniqueispracticableandeconomical.Keywords:Dotblot;Glycosyltransferase;Gene,Northernblot(dotblot),,DNARNA,,,,,,,,11.1N22(UDP2GalNAc:polypeptideN2acetylgalactosaminyltransferases2,pp2GalNAcT2EC2.4.1.41)pp2GalNAcT2DH5,,,M13PCR,M13Forward52GTAAAACGACGGCCAG23,M13Reverse52CAGGAAACAGCTATGAC23,,1200bpppGalNAc2T2cDNA1.2T25’2GAAAGACCTTCATCA2CAGCAATGGAGAAGAA,ppGalNAc2T2cDNA,31bp51.2.11.2.1.1pp2GalNAcT2cDNA1NNaOH200mMEDTA,0.4NNaOH10mMEDTA1.2.1.2pp2GalNAcT2cDNA1005min,,10min1.2.1.3(Amersco,)10min,1.2.1.42lpp2GalNAcT2cDNA,F2min,1.2.1.52SSC5min,,VECTORNIT42100cm220ml(50%,5XSSC,0.1%SDS,0.1%BSA,0.1%PVP,pH7.5PB)422h3h1.2.1.6,(50%,5SSC,0.1%SDS,0.1%BSA,0.1%PVP,10%,100g/mlDNA,),100cm210m1421.2.1.71(2SSC,0.1%SDS)42,2(0.5SSC,0.1%SDS)551.2.1.82min5min,(BSA,0.1MNaCl,0.2%Triton2100,0.1MTris,pH7.5)1.2.1.9,2(2),3730min1.2.1.103(0.1MTris,0.15MNaCl,0.3%Tween20pH7.5)37,(0.1MTris,0.1MNaCl,0.1MMgCl2,pH9.5),10ml66lNBT11lBCIP()90520062134()JPMT,February.2006,Vol.13,No.4(Semimonthly)1.2.1.11TE,1.2.21.2.2.1,:200ng/ml100ng/ml50ng/ml10ng/ml;:1/4001/8001/20001/5000;BSA:2%,2h;12,1h;,1h;1h;30min1.2.2.21.2.2.1,pp2GalNAcT2cDNA,,BSA2%3%5%1.2.2.31.2.2.2,1h30min10min;:2h1.5h50min;1h40min20min;:1h30min10min;,,15min,1.3K562NB4NIH3T3SGC7901pp2GalNAcT2NIH3T3SGC27109K562NB4;RNATRIZOLPCRpUCMixDNAmarkerladderlnvitrogen,Takara()Genetyx,OenBankBlast2MG:F:52CTCGTGCTACTCTCTCTTTC23R:52CATGTCTCGATCCCACTTAAC23330bpppGalNAc2T2:F:52GAAAGAATTAGGAAGGGTCAGAAC23R:52CTGTGTCAATGTAAACATAGCTC23668bpTRIZOLK562NB4NIH3T3SGC7901RNA,A260/A280,A260,cDNA,pp2GalNAcT2:K562NB4NIH3T3SGC7901cD2NApp2GalNAcT22MCRT2PCR22.11,10ng/ml,1/5000;,pp2GaINAcT2cDNA,BSA2%3%5%,25%BSA;,,3,23,32.2K562NB4/NIH3T3SGC7901GaINAcT2K562SGC7901NB4,NIH3T3RT2PCR,5,6,3[1]Superarray[2],,DNA,,pH7.5PB:,,pH6.5,01520062134()JPMT,February.2006,Vol.13,No.4(Semimonthly),,:,10ng/ml,122,10min,5%BSA50min,1/5000,,20min,10ml,,,SGC7901K562NB4NIH3T3cDNA,pp2GalN2AcT2,:K562SGC7901NB4,NIH3T3pp2GalNAcT2[3],,pp2GalNAcT2,,SGC7901pp2GalNAcT2mRNA:N1H3T3,,,pp2GalNAcT2K562NB4,pp2GalNAcT2,,cDNA,cDNApp2GalNAcT2RT2PCR,2MG,T22MG,T2,,,,RT2PCRNIH3T3T2,,,,cDNA,RNA,cDNARNA,cDNA,,RNA,RNA,RNA,,,cDNA,,,cDNA,,,cDNA,,:,,:[1]J.,E.F.,T..[M].:,1993:3622395.[2][3]KudoT,IkeharaY,TogayachiA,etal.Up2regulationofasetg2lycosyltransferasegenesinhumancolorectatcancer[J].Labln2vest,1998,78(7):7972811.(:2005210211)3,(,277700)[]:(HD)(SOD)(MDA)(NO)(NOS):47(HD)(HD),,SODMDANONOS,SODMDANONOS:HD(P0.01),SODMDANONOS(P0.05):,,,[];;[]R459.5[]A[]167125098(2006)0420511202InvestigationoftheHemodialysis’EffecttotheSerumOxygen2derivedFreeRadidicalsContentanditsActionZHAOChuan2guo,LIANGYong2mei(ThePeoplesHospitalofCangshancounty,Cangshan,Shandong277700,China)Abstract:ObjectiveToinvestigatetheeffectofbloodserumSODMDANONOScontentandtherapeuticaleffectinacuteorganophosphatepoisoningpatientswithHD.Methods47acuteorganophosphatepoisoningpatientsweredividedintotwogroupsrandomly:onegroupwastreatedwithHDandanotherwithoutHD.Weobservedtheoutcomeandas2sayedtheserumSODMDANONOScontent.Thehealthman’sserumSODMDANONOSwascontrast.ResultTheconsciousnesswithHDwasrecoveredearlier(P0.01),ThechangesofserumSODMDANONOShadsignificantdifference(P0.05).ConclusionHemodialysiscancleanorganophosphatepoisonutility,preventandcurepoisoningbyatropine,decreasetoxicityfreeradical,relieveprimarycellulardefectofheartandbrain.Keywords:Hemodialysis;Organophosphatepoisoning;Oxygen2derivedfreeradidicals11520062134()JPMT,February.2006,Vol.13,No.4(Semimonthly)3:(1969-),,,1990,