利用分叶顺次肝切除术建立小鼠肝脏大部切除后再生模型

52420095305:10.3724/SP.J.1008.2009.00524论著汤朝晖1,周露婷2,谢志芳3,胡以平3,杨甲梅1*,章卫平2*,吴孟超11.,2004382.,2004333.,200433[]目的:利用肝部分切除术,建立可准确量化和易于操控的小鼠肝脏再生模型,为研究肝脏再生的细胞和分子生物学机制及其病理学意义提供技术平台方法:正常成年C57BL/6小鼠经心脏灌注固定后,逐叶分离肝脏称重,计算各肝叶所占肝重的百分比;麻醉状态下,分叶顺次无菌切除小鼠肝脏的左外叶左中叶和右中叶,建立肝脏大部切除的再生模型;利用RT-PCR方法动态检测术后肝脏甲胎蛋白基因的激活表达特征结果:肝左外叶左中叶和右中叶合计约占小鼠肝脏总重量的68%,分叶顺次切除上述3个肝叶,术后动物恢复和存活状态良好,RT-PCR结果证实甲胎蛋白基因在小鼠肝脏再生中的激活表达结论:利用分叶顺次肝切除术,成功建立了小鼠的肝大部切除后再生模型,该方法可准确量化肝脏切除的程度,具有可控性强简便易行和成功率高等优点,为小鼠肝再生的研究奠定了基础[]肝解剖;肝部分切除术;肝再生[]R333.4[]A[]0258-879X(2009)05-0524-03EstablishmentofmouseliverregenerationmodelbyconsecutivepartialhepatectomyTANGZhao-hui1,ZHOULu-ting2,XIEZhi-fang3,HUYi-ping3,YANGJia-mei1*,ZHANGWei-ping2*,WUMeng-chao11.EasternHepatobiliaryHospital,SecondMilitaryMedicalUniversity,Shanghai2004382.DepartmentofPathophysiology,CollegeofBasicMedicalSciences,SecondMilitaryMedicalUniversity,Shanghai2004333.DepartmentofCellBiology,CollegeofBasicMedicalSciences,SecondMilitaryMedicalUniversity,Shanghai200433[]2008-12-29[]2009-04-10[](90608026,30772478).SupportedbyNationalNaturalScienceFoundationofChina(90608026,30772478).[],.E-mail:[e-mail]*(Correspondingauthors).Tel:[phone],E-mail:[e-mail];Tel:[phone],E-mail:[e-mail][ABSTRACT]Objective:Toestablishaquantifiableandeasilymanipulablemousemodelofliverregenerationbypartialhepatectomy,soastoprovideabasisforinvestigatingtheunderlyingcellularandmolecularmechanismsandpathophysiologicalsignificanceofliverregeneration.Methods:NormaladultC57BL/6micewerefixedbycardiacperfusion,theliverlobesweredissectedandweighed,andtheproportionsofeachlobewerecalculated.Underanestheticandsterilecondition,partialhepatectomywasperformedbyremovingtheleftlateral,leftmiddle,andrightmiddleliverlobesinturntoestablishtheregenerationmodel.Theactivationofalpha-fetoprotein(AFP)geneintheregeneratingliverwasdynamicallymonitoredbyreal-timePCRanalysis.Results:Theleftlateral,leftmiddle,andrightmiddleliverlobestogetheraccountedforapproximately70%ofthetotalliverweight.Theanimalssurvivedandlivedwellafterremovalofthethreelobesinturn.RT-PCRshowedthatAFPexpressionwasactivatedintheregeneratinglivers.Conclusion:Wehavesuccessfullyestablishedthemousemodelofliverregenerationbyconsecutivepartialhepatectomy.Thismethodcanquantifytheheptectomyandiseasytoperform,whichlaysafoundationforstudyingliverregenerationinmice.[KEYWORDS]liveranatomy;partialhepatectomy;liverregeneration[AcadJSecMilMedUniv,2009,30(5):524-526],HigginsAnderson1931,[1],,,,,5.,.525,[2],,11.1动物C57BL/6Jackson,SPF6~10,,20~30g1.2肝脏的分叶解剖,4%(8l/g),,;,,,,20ml0.1mol/LPBS(pH7.4),20ml4%,,;,1.3肝大部切除术,4%,,,,,,,1,,,,1,,(70%),,05ml5%,37,1.4甲胎蛋白mRNA荧光定量RT-PCR检测,-80TRIzol(Invitrogen)RNA,SuperScript-Oligo(dT)(Invitrogen)cDNA;SYBRgreen(AFP)PCR(Mastercyclereprealplex,Eppendorf),Ct,36B4,AFPmRNA[3]AFPmRNA,244872hAFPmRNA1.5统计学处理t,P0.0522.1小鼠肝脏的解剖和分叶特征,;,(1),,,,,1/4,1/2,,3,,20C57BL/6,:37.12%,9.46%,19.40%,13.46%,11.48%,7.3%,68%1Fig1ObservationofmouseliveranatomyinvivoandinvitroA:Invivoinspection;B:Diaphragmaticview;C:Visceralview.LLL:Leftlaterallobe;LML:Leftmiddlelobe;RML:Rightmiddlelobe;RUL:Rightupperlobe;RLL:Rightlowerlobe;CL:Caudatelobe.FL:Falciformligament;GB:Gallbladder;ST:Stomach;SP:Spleen2.2分叶顺次肝切除术建立小鼠的大部肝切除后再生模型,,,52620095,30,20,2,1,1,90%2.3肝切除后再生肝组织AFP表达定量分析AFP,[3]AFPRT-PCR,24hAFP,48h,72h8(2);48h72hAFP24h,(P0.05,P0.01)2AFPmRNAFig2ActivatedexpressionofAFPmRNAinmouseliverafterpartialhepatectomy*P0.05,**P0.01vs24hgroup;n=4,xs3,,,,,C57BL/6,,70%[4-5],,RT-PCRAFP,,,90%95%97%[6],,,90%[7];,95%;,97%,,,[8],,:(1),;,,,(2),(3),,,(),,,,(4)(5),20%,90%[9][][1]HigginsGM,AndersonRM.Experimentalpathologyoftheliver:restorationoftheliverofthewhiteratfollowingpartialsurgicalremoval[J].ArchPathol,1931,12:186-202.[2]MartinsPN,TheruvathTP,NeuhausP.Rodentmodelsofpartialhepatectomies[J].LiverInt,2008,28:3-11.[3]XieZ,ZhangH,TsaiW,ZhangY,DuY,ZhongJ,etal.ZincfingerproteinZBTB20isakeyrepressorofalpha-fetoproteingenetranscriptioninliver[J].ProcNatlAcadSciUSA,2008,105:10859-10864.[4]RodrguezG,LorenteL,DurnHJ,AllerMA,AriasJ.A70%hepatectomyintheratusingamicrosurgicaltechnique[J].IntSurg,1999,84:135-138.[5]FaustoN,RiehleKJ.Mechanismsofliverregenerationandtheirclinicalimplications[J].JHepatobiliaryPancreatSurg,2005,12:181-189.[6]MadrahimovN,DirschO,BroelschC,DahmenU.Marginalhepatectomyintherat:fromanatomytosurgery[J].AnnSurg,2006,244:89-98.[7]EmondJ,Capron-LaudereauM,MeriqqiF,BernuauJ,ReynesM,HoussinD.Extentofhepatectomyintherat.Evaluationofbasalconditionsandeffectoftherapy[J].EurSurgRes,1989,21:251-259.[8]MartinsPN,NeuhausP.Hepaticlobectomyandsegmentectomymodelsusingmicrosurgicaltechniques[J].Microsurgery,2008,28:187-191.[9]NikfarjamM,Malcontenti-WilsonC,FanartzisM,DaruwallaJ,ChristophiC.Amodelofpartialhepatectomyinmice[J].JInvestSurg,2004,17:291-294.[],