HLA分型技术.pptx

1 HLAHLA分型技术分型技术上海交通大学医学院彭奕冰 主任技师2Major Histocompatibility ComplexMajor Histocompatibility Complex(MHC)(MHC)What is MHC?What is MHC?HLAHLAH-2H-2Minor histocompatibility antigensMinor histocompatibility antigens3SignificanceoftheMHCSignificanceoftheMHCrole in immune responserole in immune responserole in organ transplantationrole in organ transplantationrole in predisposition to diseaserole in predisposition to disease5GeneticbarrierstotransplantationGeneticbarrierstotransplantationautologous:in the same autologous:in the same individual(autograft)individual(autograft)isologous:between isologous:between genetically Identical genetically Identical individuals(isograft),individuals(isograft),i.e.i.e.,identical twins(inbred identical twins(inbred animals)animals)homologous:between homologous:between individuals of the same individuals of the same species(allograft)species(allograft)heterologous:between heterologous:between individuals different species individuals different species(xenograft)(xenograft)6PrinciplesoftransplantationPrinciplesoftransplantation7MinorhistocompatibilityantigensMinorhistocompatibilityantigensandgraftsurvivalandgraftsurvivalminor histocompatibility antigens also cause rejectionThe rejection time is variable but longer than that for major histocompatibility antigenThey have additive effects8Graftversushost(GVH)Graftversushost(GVH)diseasedisease9GVHdiseaseinhumansGVHdiseaseinhumans10ThehumanMHCgenesThehumanMHCgenes11ThemouseMHCgenesThemouseMHCgenes12PolymorphismofMHCantigensPolymorphismofMHCantigens(basedonphenotype)(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正式命名显示多态性的HLA基因座位有基因座位有31个，共个，共2320个等位基因。个等位基因。图中所示为常见基因座位的图中所示为常见基因座位的等位基因数。等位基因数。13TheinheritanceofMHCgenesTheinheritanceofMHCgenes14Crossingoverresultsinnewhaplotypes15MHCproductsexpressedoncellsMHCproductsexpressedoncellsIf Jack and Jill have four children;Bo,Kim,Mo and LeeTheyll all inherit antigens of the parental MHCOft their haplotypes will be of the father or motherUnless during meiosis,a crossover should occur16Class-I expressed on all nucleated cells in man,and also on erythrocytes in mice.Class-II expressed primarily on antigen presenting cells(dendritic cells,macrophages and B cells,etc.)DifferentialexpressionofMHCantigens17Alloreactivity of T cells:Alloreactivity of T cells:MLR and CTL generationMLR and CTL generationCD4+TH1CD8+CTLCD8+preCTL18Alloreactivity of T cellsAlloreactivity of T cellsPositiveSelectionThymusAlloreaction(MLR)Proliferation and Differentiation19InflammationlysisADCClysisIL2,IFN TNF,NO2IL2,IL4,IL5IL2,TNF,IFN rejectionMechanismsofgraftrejectionMechanismsofgraftrejection20TempoofrejectionreactionTempoofrejectionreactiontypeofrejectiontimetakentimetakencausecausechronicchronicmonths-yearsmonths-yearsunclear causes:cross reactive Ab,unclear causes:cross reactive Ab,immune complexes,slow cellular immune complexes,slow cellular reaction,tolerance breakdown,reaction,tolerance breakdown,disease recurrencedisease recurrenceaccelerateddaysreactivation of sensitized T cells(secondary response)acutedays-weeksprimary activation of T cellshyperacuteminutes-hourspreformed anti-donor antibodies and complement21A,B&DRmatchingandgraftA,B&DRmatchingandgraftsurvivalsurvival22Removal of T cells from marrow graftRemoval of T cells from marrow graft MagnetMagnetic antibodies23HLAanddiseaseassociationHLAanddiseaseassociationDiseaseDiseaseAssociated Associated allelesallelesFrequency inFrequency inRelative Relative riskriskpatientspatientscontrolcontrolAnkylsoing spondylitisAnkylsoing spondylitisReiters diseaseReiters diseaseAcute anterior uveitisAcute anterior uveitisPsoriasis vulgarisPsoriasis vulgarisDermatitis herpetiformisDermatitis herpetiformisB27B27B27B27B27B27CW6CW6DR3DR39 979795252878785859 99 99 93333262687.487.437.037.010.410.413.313.315.415.424HLA抗原检测法抗原检测法25微量细胞毒试验微量细胞毒试验原理原理当特异性抗体与淋巴细胞表面当特异性抗体与淋巴细胞表面HLA分子分子结合，激活补体，使细胞溶解。在显微镜下结合，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。可见细胞被活性染料着色。淋巴细胞只表达淋巴细胞只表达HLA I分子，如欲测分子，如欲测定定HLA II类分子，需用细胞。类分子，需用细胞。26微量细胞毒试验微量细胞毒试验 HLA I类分型法类分型法方法方法1.加板：加板：Terasaki板中加入特异性抗板中加入特异性抗HLA I类分子类分子抗体。抗体。2.分离分离PBMC。制成浓度为制成浓度为1 106/ml的细胞悬液。的细胞悬液。3.每孔内加入每孔内加入1 l细胞悬液。细胞悬液。4.室温培育室温培育30min。5.1 l兔补体。室温培育兔补体。室温培育60min。6.每孔内加入每孔内加入5 l 5%伊红水溶液。室温伊红水溶液。室温5min。7.每孔内加每孔内加37%甲醛固定。甲醛固定。8.倒置相差显微镜下观察结果。倒置相差显微镜下观察结果。27微量细胞毒试验微量细胞毒试验 HLA II类分型法类分型法与与HLA类分型法区别类分型法区别1须用富含细胞的淋巴细胞悬液。须用富含细胞的淋巴细胞悬液。2抗体与细胞培育时间为抗体与细胞培育时间为1h。3加补体后培育时间为加补体后培育时间为2h。28微量细胞毒试验微量细胞毒试验应用实践应用实践 个体个体HLA分型可用于移植前供者和受者分型可用于移植前供者和受者配型，亲子鉴定，法医鉴定等。配型，亲子鉴定，法医鉴定等。29交叉配型交叉配型 用于测定受者血清中受含有抗供者用于测定受者血清中受含有抗供者HLA的的抗体。如受者血清中具有抗供者红细胞血型抗体。如受者血清中具有抗供者红细胞血型抗原抗体和抗原抗体和/或抗白细胞抗原抗体，会引起超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定，以及测定患者血清中是否具有抗供者HLA抗体。抗体。30交叉配型交叉配型方法方法1.分离供体分离供体PBMC。2.分离受者血清，作分离受者血清，作1：2，1：4和和1：8稀释。加稀释。加入入Terasaki板，每孔板，每孔1 l。其余步骤同微量细胞毒试验。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如结果为阳性，表明受者血清中具有抗供者HLA抗体。抗体。31细胞学分型法细胞学分型法 细胞学分型法已被血清学和分子生物学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方法代替。单相混合淋巴细胞培养实验还在使用。在使用。32分子生物学技术为基础的分子生物学技术为基础的HLA分型法分型法原理原理HLA的多态性是由基因中核苷酸序列突变引起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，变异集中在几个大多是相同的，变异集中在几个DNA片段中。片段中。在这些片断中，不同的等位基因具有特定的序在这些片断中，不同的等位基因具有特定的序列。测定这些片段的列。测定这些片段的DNA序列就可以确定等序列就可以确定等位基因位基因。33 变异片段变异片段 变异片段变异片段 变异片段变异片段34 a b c dac Aac ATCGGTCGGTCAAGGTCAAGGCCTATCCCTATCGATTGCGTAGGCGATTGCGTAGGC bd ATCGGTbd ATCGGTCAAGCAAGGCCTATCGGCCTATCGATTGATTGCGTAGGCCGTAGGC ac Aac ATCGGTCGGTCAAGGTCAAGGCCTATCCCTATCGATTGCGTAGGCGATTGCGTAGGCad Aad ATCGGTCGGTCAAGGCCTATCGTCAAGGCCTATCGATTGATTGCGTAGGCCGTAGGC bc ATCGGTbc ATCGGTCAAGCAAGG GCCTATCCCTATCGATTGCGTAGGCGATTGCGTAGGC35根据片段组合指定等位基因根据片段组合指定等位基因acacacacbcbcad ad bdbdacacefefghghefefmlmljkjkoxoxrtrtwqwqsvsvzpzp