HLA分型技术ppt课件

,HLA分型技术,上海交通大学医学院彭奕冰主任技师,1,MajorHistocompatibilityComplex(MHC),WhatisMHC?HLAH-2Minorhistocompatibilityantigens,2,SignificanceoftheMHC,roleinimmuneresponseroleinorgantransplantationroleinpredispositiontodisease,3,Progressinorgantransplantation,4,Geneticbarrierstotransplantation,autologous:inthesameindividual(autograft)isologous:betweengeneticallyIdenticalindividuals(isograft),i.e.,identicaltwins(inbredanimals)homologous:betweenindividualsofthesamespecies(allograft)heterologous:betweenindividualsdifferentspecies(xenograft),5,Principlesoftransplantation,6,Minorhistocompatibilityantigensandgraftsurvival,minorhistocompatibilityantigensalsocauserejectionTherejectiontimeisvariablebutlongerthanthatformajorhistocompatibilityantigenTheyhaveadditiveeffects,7,Graftversushost(GVH)disease,8,GVHdiseaseinhumans,9,ThehumanMHCgenes,10,ThemouseMHCgenes,11,PolymorphismofMHCantigens(basedonphenotype),HLA基因系统的多态性及一些主要基因座位的等位基因数正式命名显示多态性的HLA基因座位有31个，共2320个等位基因。图中所示为常见基因座位的等位基因数。,12,TheinheritanceofMHCgenes,13,Crossingoverresultsinnewhaplotypes,14,MHCproductsexpressedoncells,IfJackandJillhavefourchildren;Bo,Kim,MoandLeeTheyllallinheritantigensoftheparentalMHCOfttheirhaplotypeswillbeofthefatherormotherUnlessduringmeiosis,acrossovershouldoccur,15,Class-Iexpressedonallnucleatedcellsinman,andalsoonerythrocytesinmice.,Class-IIexpressedprimarilyonantigenpresentingcells(dendriticcells,macrophagesandBcells,etc.),DifferentialexpressionofMHCantigens,16,AlloreactivityofTcells:MLRandCTLgeneration,CD4+TH1,CD8+CTL,CD8+preCTL,17,AlloreactivityofTcells,PositiveSelection,Thymus,Alloreaction(MLR),ProliferationandDifferentiation,18,Inflammation,lysis,ADCC,lysis,IL2,IFN,TNF,NO2,IL2,IL4,IL5,IL2,TNF,IFN,rejection,Mechanismsofgraftrejection,19,Tempoofrejectionreaction,20,A,B&DRmatchingandgraftsurvival,21,RemovalofTcellsfrommarrowgraft,Magneticantibodies,22,HLAanddiseaseassociation,23,HLA抗原检测法,24,微量细胞毒试验,原理当特异性抗体与淋巴细胞表面HLA分子结合，激活补体，使细胞溶解。在显微镜下可见细胞被活性染料着色。淋巴细胞只表达HLAI分子，如欲测定HLAII类分子，需用细胞。,25,微量细胞毒试验HLAI类分型法,方法加板：Terasaki板中加入特异性抗HLAI类分子抗体。分离PBMC。制成浓度为1106/ml的细胞悬液。每孔内加入1l细胞悬液。室温培育30min。1l兔补体。室温培育60min。每孔内加入5l5%伊红水溶液。室温5min。每孔内加37%甲醛固定。倒置相差显微镜下观察结果。,26,微量细胞毒试验HLAII类分型法,与HLA类分型法区别1须用富含细胞的淋巴细胞悬液。2抗体与细胞培育时间为1h。3加补体后培育时间为2h。,27,微量细胞毒试验,应用实践个体HLA分型可用于移植前供者和受者配型，亲子鉴定，法医鉴定等。,28,交叉配型,用于测定受者血清中受含有抗供者HLA的抗体。如受者血清中具有抗供者红细胞血型抗原抗体和/或抗白细胞抗原抗体，会引起超急排异反应。所以移植前应作红细胞血型测定，以及测定患者血清中是否具有抗供者HLA抗体。,29,交叉配型,方法1.分离供体PBMC。2.分离受者血清，作1：2，1：4和1：8稀释。加入Terasaki板，每孔1l。其余步骤同微量细胞毒试验。如结果为阳性，表明受者血清中具有抗供者HLA抗体。,30,细胞学分型法,细胞学分型法已被血清学和分子生物学方法代替。单相混合淋巴细胞培养实验还在使用。,31,分子生物学技术为基础的HLA分型法,原理HLA的多态性是由基因中核苷酸序列突变引起的。同一基因的不同等位基因之间核苷酸序列大多是相同的，变异集中在几个DNA片段中。在这些片断中，不同的等位基因具有特定的序列。测定这些片段的DNA序列就可以确定等位基因。,32,变异片段变异片段变异片段,33,abcd,acATCGGTCAAGGCCTATCGATTGCGTAGGC,bdATCGGTCAAGGCCTATCGATTGCGTAGGC,acATCGGTCAAGGCCTATC