HLA分型技术PPT课件

1、1Major Histocompatibility Complex(MHC)What is MHC?HLAH-2Minor histocompatibility antigens第1页/共35页2Significance of the MHC role in immune response role in organ transplantation role in predisposition to disease第2页/共35页3Progress in organ transplantation第3页/共35页4Genetic barriers to transplantationautol

2、ogous: in the same individual (autograft)isologous: between genetically Identical individuals (isograft), i.e., identical twins (inbred animals)homologous: between individuals of the same species (allograft)heterologous: between individuals different species (xenograft)第4页/共35页5Principles of transpl

3、antation第5页/共35页6Minor histocompatibility antigensand graft survivalminor histocompatibility antigens also cause rejectionThe rejection time is variable but longer than that for major histocompatibility antigenThey have additive effects第6页/共35页7Graft versus host (GVH) disease第7页/共35页8GVH disease in

4、humans第8页/共35页9The human MHC genesclass IIclass IDPDQDRBCABCADPDQDR122211931 221 3第9页/共35页10The mouse MHC genesIDKAEclass IIclass Iclass Iclass III(C4, factor-B,TNF, etc.)第10页/共35页11Polymorphism of MHC antigens (based on phenotype) 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2 320个等位基因。 图中所示为常见

5、基因座位的等位基因数。第11页/共35页12The inheritance of MHC genesJackJillDPDQDRBCA1234BoKimMoLee13412342第12页/共35页13Crossing overresults in new haplotypes第13页/共35页14MHC products expressed on cellsIf Jack and Jill have four children; Bo, Kim, Mo and LeeTheyll all inherit antigens of the parental MHCOft their haploty

6、pes will be of the father or motherUnless during meiosis, a crossover should occur第14页/共35页15 Class-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.)Differential expre

7、ssion of MHC antigens第15页/共35页16Alloreactivity of T cells:MLR and CTL generationCD4+TH1CD8+CTLCD8+preCTL第16页/共35页17Alloreactivity of T cellsT CELLT CELLDRW11DRW17PositiveSelectionThymusAlloreaction(MLR)Proliferation and Differentiation第17页/共35页18InflammationlysisADCClysisIL2, IFN TNF, NO2IL2, IL4, I

8、L5IL2, TNF, IFN rejectionMechanisms of graft rejection第18页/共35页19Tempo of rejection reactiontype of rejectionaccelerateddaysreactivation of sensitized T cells (secondary response)acutedays-weeksprimary activation of T cellshyperacuteminutes- hourspreformed anti-donor antibodies and complement第19页/共3

9、5页20A, B & DR matching and graft survival第20页/共35页21Removal of T cells from marrow graft MagnetMagnetic antibodies第21页/共35页22HLA and disease association第22页/共35页23HLA抗原检测法第23页/共35页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HLA I分子，如欲测定HLA II类分子，需用细胞。第24页/共35页25微量细胞毒试验 HLA

10、 I类分型法方法1.加板：Terasaki板中加入特异性抗HLA I类分子抗体。2.分离PBMC。制成浓度为1 106/ml的细胞悬液。3.每孔内加入1 l细胞悬液。4.室温培育30min。5.1 l兔补体。室温培育60min。6.每孔内加入5 l 5%伊红水溶液。室温5min。7.每孔内加37%甲醛固定。8.倒置相差显微镜下观察结果。第25页/共35页26微量细胞毒试验 HLA II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。第26页/共35页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

11、医鉴定等。第27页/共35页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。第28页/共35页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1 l。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。第29页/共35页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。第30页/共35页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