【doc】 胆总管狭窄对大鼠肝组织BR-UDPGT基因表达的影响

【doc】 胆总管狭窄对大鼠肝组织BR-UDPGT基因达的影响 胆总管狭窄对大鼠肝组织BR-UDPGT基因 表达的影响 内蒙古医学院2005年9月第27卷第3期?165? 年医学本科毕业后从事外科工作至今,目前主要从事普通外科的临床’… r=1治疗与研究.主持多项国家及自治区级自然科学基金项目,两次入选内 副主任委员,中华医学会内蒙古普通外科分会常务委员,内蒙古和呼和???霉,l 浩特市医疗事故鉴定专家库成员.I’?_ ? 论着? 文章编号:1004—2113(2005)03-0165.05 EFFECTOFCBDSTRICTUREONTHEEXPRESSIONOFTHE BILIRUBINUDP—GLUCURONOSYLTRANSFERASE INRATLIVERTISSUES ZHANGRui—ming,GAOQuan—rong,SHENTao,ZHOUXiao—si (AffiliatedHospital,InnerMongoliaMedicalCollege,Hohhot010050China) Abstract:ThisessayisaimedtostudythechangeoftheexpressionofBR—UDPGT.Theelevated concentrationofunconjugatedbilirubin(UCB)inbileplaysacrucialroleinthepathogenesisofPS.ItisweII knownthatthebileductstrictureisoneofthefactorsrelatedtopathogenesisofPS.InthePSanimalmodel inducedbycommonbileduct(CBD)stricture,neitherbiliaryinfectionnorenhancedactivityof/]--glucuronidase (p—G)wereidentified,whereastheconcentrationofUCBinbilewasmarkedlyelevatedandPSformed. Therefore,inordertofurtherstudythepathogenesisofPS,itisnecessarytoinvestigatethechangeofBR—— UDPGTafterCBDstricture.AdultmaleWistarratsweredividedintocontrolandseveralCBDstricture groups.TheexpressionofBR--UDPGTwasinvestigatedbyNorthernblotandmeasuringtheactivityofBR— UDPGT.Duringtheperiodof12hoursto14daysafterCBDstricture,theexpressionofBR—UDPGTin mRNAwasalmostthesameasthatofthecontrolgroup.ButtheactivityofBR--UDPGTwasreduced12hoIrs afterCBDstricture,from14.82vmol/g(protein)/h士 0.45~umol/g(protein)/hto11.67~umol/g(protein)/ h士1.26~umol/g(protein)/h,(i士 s.bothn=5,P<O.O1).Anditbecamemuchlowerwiththedurati0nof CBDstricture.CBDstricturedecreasedtheactivityofBR—UDPGTandredu cedtheabilityoflivertissuest0 transferUCBintoCB.ThelevelaffectedbyCBDstricturewasnotthetranscrip tionofBR—UDPGT.Other stepsaftergenetranscriptionmaybeinvolved.ThedecreasedactivityofBR-- UDPGTmaybeoneofthecauses ofelevatedUCBconcentrationafterCBDstricture. Keywords:livertissues;activityofenzyme;bilirubin;geneexpression 中图分类号:R575.7文献标识码:A 收稿日期:2005?07?25;修回日期:2005.08.26 基金项目:国家自然科学基金资助项目(39670710). ?166? Unconjugatedbilirubin(UCB)playsan importantroleintheoccurenceanddevelopment ofpigmentgallstones(PS).Bilirubinisconverted intoconjugatedbilirubinintheliver,whichis catalysedbythebilirubin’——UDPglucuronosyl-- transferase(BR—UDPGT)andthenexcreted intobile.Pigmentgallstons(PS)contain considerableamountsofunconjugatedbilirubin (UCB)intheformofcalciumbilirubinateand/or bilirubinpolymers.Theelevatedconcentrationof UCBinbileplaysacrucialroleinthe pathogenesisofPS.Sincemorethan98ofbile pigmentsareexcretedasconjugatesofbilirubin, thesourceofthisUCBneedstobeidentified. MakisuggestedUCBcamefromthehydrolysisof conjugatedbilirubin,whichwasmediatedby(一 G)ofbacterialorigin.InthePSanimalmodel inducedbycommonbileduct(CBD)stricture, neitherbiliaryinfectionnorenhancedactivityof —— Gwereidentified,whereastheconcentrationof U『CBinbilewasmarkedlyelevatedandPS formed.Theprimaryculturedhepatocytes, isolatedfromtheliverofWistarratswithCBD stricture,possessedloweredabilitytotranfer UCBintoCB.Thepurposeofthispaperisto investigate expression theeffectofCBDstrictureon oftheBR——UDPGT. 1Materialsandmethods 1.1Experimentalanimalsmodels MaleWistarrats(200,350g)were purchasedintheDepartmentofExperimental Animal,BeijingMedicalUniversityandrandomly devidedintocontrolandseveralCBDstricture groups.Underpentobarbitalanesthesia(30mg/ kgbodyweight,intraperitoneaUy),therats underwentsurgeryinthemorning.Inthecontrol group,apieceofliverfromtheleftlaterallobe wascutoff,whereasCBDwasligated incompletelyintheCBDstricturegroups.The ratsofCBDstricturegroupshadfreeaccessto foodanddrinkingwateraftersurgeryandthey werecutoffapieceofliverfromleftlaterallobe ActaAcadMedNeiMongolSep.2005Vo1.27No.3 undersimilaranesthesiainthemorning12hours and1,3,5,7,10,14daysafterfirstsurgery. 1.2NorthernBlotHybridization TotalRNAwaspreparedfromlivertissues byacidguanidiniumthiocyanate’’phenol-’ chloroformextraction.TheRNAwasdenatured informamide,formaldehydeandelectrophoresed ina1.0agarosedenaturedwithformaldehyde. RNAwastransfusedtoasheetofnitroceUulose filterbycapillaryelution.Thefilterwasdredat 80~Cfor2hours.Aphotographwastakento performimageanalysisforsampleamount comparison(IOD).Prehybridizati0nand hybridizationwerethenperformedat42~Cfor24 hourswithB—UDPGT1cDNAprobethathad dCTP. beenrandomlyprimerlabeledwith.P— Anautoradiographywasperformedbyexposing thefiltertoX—rayfilmat一70?for72hours, usinganintensifingscreen.Theexpressionlevel wasthenexaminedthroughtheimageanalysis (IOD). 1.3TheactivityofBR—UDPGTofliverwas asseyedbythemethodintroducedbyM.Black Iiverhomogenateswerepreparedand homogenate?——digitoninmixtureswereincubated withbilirubinandUDPGAatpH7.4,37?for30 min.Bilirubinwastransferedtoconjugated bilirubinduringtheincubation.Conjugated bilirubincouldbecoupledselectivelywiththe diazoniumsaltofethylanthranilateatpH2.7. Afterthediazotisationreaction,extractionofthe azo--pigmentswascarriedoutwithamethyln-- —butylacetatemixture(17/3,v/ propylketone—n pigment— v).Thencentrifuged,theazo— containingupperlayerwaspipettedoffandit’s E530valuemeasured,thenthemolarsofazo— pigmentinextractionorganicsolventwas calculated.Theoretically,formedbilirubin conjugatedincludeI,theazo—pigmentin extractionorganicsolventand?.theazo— pigmentinwaterphaseand??thebilirubin conjugatedhydrolysedbyB—glucuronidaseof liverorigin.Becausethepartitioncoefficientof 内蒙古医学院2005年9月第27卷第3期 azo--pigmentbetweentheextrationorg’anicphase andwaterphaseinthisreactionsystemwasmore than1000.?couldbeneglected.Accordingto theresultsmentionedinthispaper(1.4),the extentofinterferenceofliveroriginalB— glucuronidaseontheCBformedwasabout0, 5,SO?couldalsobeneglected.Therebythe azo’——pigmentinextractionorganicsolventcould reflecttheactivityofBR--UDPGT. 1.4ActivityofliveroriginalB—glucuronidase wasassayedaccordingtothemethodintroduced byFishmanusingphen0lphthannasthe substrate.Assaywascarriedoutwithsameliver homogenateandincubationtimeformeasurement ofBR—UDPGT.Phenolphthalincolourwas developedbyaddingtheglycinebuffer(pH11.2) totesttheactivityoff}-Gunderthisspecific reactioncondition.Themolarsofphenolphthalin releasedwascalculated. 1.5Statisticalanalysis:Comparisonbetween controlgroupandCBDstricturegroupsbymeans oft—test. 2Results ?167? 2.1Northernblothybridization:Asthefilm showed,theexpressionofBR—UDPGTin mRNA12hoursto14daysafterCBDstricture wasalmostthesameasthatofthecontrolgroup. 2.2OntheseventhdayafterCBDstricture,the activitiesofBR—UDPGTandB—glucuronidase (BG)ofsameliverhomogenateswereassayed, theextentofinterferenceofliveroriginalB— glueuronidase(8G)onthecoagulatedCBwas about0,5,whichcouldbeneglected.The molarsofazo’——pigmentinextractionorganic solventcould(really)reflecttheactivityofB— UDPGT.Therebytheactivitiesofliver homogenateswerenotassayedatothergroups afterCBDstricture.(Seetable1.) 2.3ActivityofBR—UDPGTwasreduced12 hoursafterCBDstricture.Anditbecamemuch lowerwiththedurationofCBD.(Seetable2.) Tab.1Theinterferenceexperimentontheinfluenceof8一 Gactivityinlivertissuesontheassayof BR—UDPGTactivity[(~mol/g(protein)/h] notela.RectifiedactivityofBR--UDPGT=assayedactivityofBR—UDPGT +activityofB—G b.InterferenceofB—G()=(activityof~]--G/rectifiedactivityofBR--UDPG T)×100 Tab.2ActivityofBR--UDPGTofcontrolandCBDstricturegroups[-ttmol/g( protein)/h] ?168? 3Discussion BR—UDPGTisanimportantpartofUGT family,whichfavourstheexcretionofbilirubin bytransferingbilirubintoconjugatedbilirubin. “Calciumbilirubinate”and/orbilirubinpolymer aremaincomponentsandinsolublepartsofPS. Althoughmanyfactorswereinvolvedinthe pathogenesisofPS,nodoubttheincreasedUCB wasanimportantcause.MakisuggestedUCB camefromthehydrolysisofconjugatedbilirubin, whichwasmediatedbyB—glucuronidaseof bacterialorigin.InthePSanimalmodelinduced bycommonbileduct(CBD)stricture,neither biliaryinfectionnorenhancedactivityofB—G wereidentified,whereastheconcentrationof UCBinbilewasmarkedlyelevatedandPS formed.ItwasdifficulttoexplainitbyMaki’ theory.Thisexperimentwasdesignedtofurther investigatethemechanismofelevatedUCBinbile whenbiliarystrictureexisted. ThemethodformeasuringtheactivityofBR — UDPGT,usedinthisexperiments,wasto assaytheformedbilirubinconjugatedcatalysedby BR—UDPGT.Theoretically,afterincubationof liverhomogenatewithbilirubinandUDPGA,the bilirubinconjugatedformedshouldbetheresults UDPGTtransferingbilirubintobilirubin ofBR— conjugatedandB—glucuronidasehydrolysing bilirubinconjugatedintobilirubinandUDPGA. M.Blacketa1.reportedthattheproductionof bilirubinconjugatedcouldnotbeincreasedby addingtheinhibitorof8一glucuronidaseto reactionsystem.Thisresultindicatedthatthe influenceof8一glucuronidaseontheassayofBR — UDPGTwasminimalundertheconditionfor assayingBR—UDPGT.However,therabbitPS modelwithbiliarystrictureandbiliaryinfection reportedbyXiaoLu—jia,theactivityofliver ActaAcadMedNeiMongolSep.2005Vo1.27No.3 originalB—glucuronidasebegantoascendfrom theprelithogenesis(1,7days)andthenmaintain nearlyaplateauatrelativelyhighleve1.Inorder toeliminatethepossibilityofhydrolysisof bilirubinconjugatedbyliveroriginalB— glucuronidaseandresultinginafalsehoodof decreasedactivityofBR——UDPGT,theactivities ofBR—UDPGTandB—glucuronidasewere assayed7daysafterCBDstricturewithsameliver homogenateundertheconditionforBR—UDPGT assay.Theresultsindicatedtheinfluenceof8一 glucuronidaseontheassayofBR—UDPGTwas inconsiderableandcouldbeneglected(tab.1). Therefore,astab.2showed,theactivityofBR— UDPGTwasindeedreducedafterCBDstricture. ShiHai—kun’sinvestigationshowedthatthe primaryculturedhepatocytes,isolatedfromthe IiverofWistarratswithCBDstricture,possessed loweredabilitytotransferUCBintoCBand explainedthenegativeeffectofCBDstrictureon theactivityofBR—UDPGTinanotheraspect. Whichstepwasinvolvedandresultedinthe reducedactivityofBR?——UDPGTafterCBD stricture?TheexpressionofBR—UDPGT mRNAduring12hto14daysafterCBDstricture wasnotweakerthanthatofthecontrolgroup. However,theactivityofBR——UDPGTwas obviouslyreducedwiththedurationofCBD stricture.Itindicatedthatthelevelaffectedby CBDstricturewasnotthetranscriptionofBR— UDPGT.Otherstepsaftergenetranscription maybeinvolved.Furtherinvestigationis necessarytomakesureoftheexactmechanismof thisphenomenon.ThedecreasedactivityofBR—— UDPGTmaybeoneofcausesofelevatedUCB concentrationinbileandthenfavouredthe lithocenosisafterCBDstricture. (下转第172页) ?172?ActaAcadMedNeiMongolSep.2005Vo1.27No.3 EFFECTSOFDBDTOENDOTHELIALCELLSAND HEMOPoIETICCELLSINMICEMARRoWONTHE PRoLIFERATIONANDDIFFERENTIATION WUYan,BILi—fu,GONGIi (DepartmentofHistologyandEmbryology.InnerMongoliaMedicalCollege,Hohhot010059China) Abstract:Objective:Toinvestigatetheactionofdangguibuxuedecoction(DBD)onthe proliferationanddifferentiationofendothelialcellsandbonemarrowhemopoieticstemcells.Methods: ByculturingtheendothelialcellsandaddingDBDwithdifferentconcentrationtothemedium,ELISA analysis,Westernblotting,Flowcytometryanalysisandhemopoieticcolonyassaywereused.Results: DBDcanpromotetheendothelialcellsfromG1phasetOSphaseandtheproductionofcytokinein endothelialcells(If——6andGM--CSF,etc.),butcannotincreasetheproliferationanddifferentiation ofhemopoieticceilsinmicemarrow.Conclusion:Invitro,theeffectsofDBDontheproliferationand differentiationofhemopoieticcellsdependonregulationofsecretingcytokinesinendothelialcellsby DBD. Keywords:danggui;huangqi;endothelialcells;cytokines;hemopoiesis 坐鲁坐坐坐j坐坐啦坐坐?;}坐坐坐坐坐坐坐坐坐k (上接第168页) 胆总管狭窄对大鼠肝组织BR--UDPGT基因表达的影响 张瑞明,高权荣,沈韬,周孝思 (1.内蒙古医学院附属医院,内蒙古呼和浩特010050;2.北京大学第三医院普外科) 摘要:为研究胆总管狭窄时肝组织BR—UDPGT基因表达的改变.本研究用雄性Wistar大鼠不完全结扎 胆总管造成胆总管狭窄动物模型采用Northern印迹杂交和测定BR—UDPGT活性的方法,研究胆总管狭窄对 肝组织BR—UDPGT基因表达的影响.结果显示:在胆总管狭窄后12h,14d的不同时间段,肝组织BR— UDPGT在mRNA水平均未见基因表达较正常对照组降低.但BR—UDPGT酶活性在胆总管狭窄后l2h后即 开始下降,且随狭窄时间的延长BR—UDPGT活性逐渐下降.胆总管狭窄确可引起肝组织BR—UDPGT的活性 下降.作用机制不在BR—UDPGT的基因转录水平,可能在转录后的其他环节上.BR—UDPGT活性下降可能 是胆总管狭窄后胆汁中的UCB浓度增高的原因之一. 关键词:肝组织;酶活性;胆红素;基因表达