,*,单击此处编辑母版标题样式,PPT课件,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,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,PPT课件,HLA分型技术上海交通大学医学院1PPT课件,Major Histocompatibility Complex(MHC),What is MHC?,HLA,H-2,Minor histocompatibility antigens,2,PPT课件,Major Histocompatibility Compl,Significance of the MHC,role in immune response,role in organ transplantation,role in predisposition to disease,3,PPT课件,Significance of the MHCrole in,Progress in organ transplantation,4,PPT课件,Progress in organ transplantat,Genetic barriers to transplantation,autologous: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),5,PPT课件,Genetic barriers to transplant,Principles of transplantation,6,PPT课件,Principles of transplantation6,Minor histocompatibility antigensand graft survival,minor histocompatibility antigens also cause rejection,The rejection time is variable but longer than that for major histocompatibility antigen,They have additive effects,7,PPT课件,Minor histocompatibility antig,Graft versus host(GVH)disease,8,PPT课件,Graft versus host(GVH)diseas,GVH disease in humans,9,PPT课件,GVH disease in humans9PPT课件,The human MHC genes,10,PPT课件,The human MHC genes10PPT课件,The mouse MHC genes,11,PPT课件,The mouse MHC genes11PPT课件,Polymorphism 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。图中所示为常见基因座位的等位基因数。,12,PPT课件,Polymorphism of MHC antigens(,The inheritance of MHC genes,13,PPT课件,The inheritance of MHC genes13,Crossing overresults in new haplotypes,14,PPT课件,Crossing overresults in new h,MHC products expressed on cells,If Jack and Jill have four children;Bo,Kim,Mo and Lee,Theyll all inherit antigens of the parental MHC,Oft their haplotypes will be of the father or mother,Unless during meiosis,a crossover should occur,15,PPT课件,MHC products expressed on cell,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 expression of MHC antigens,16,PPT课件,Class-I expressed on all nucle,Alloreactivity of T cells:,MLR and CTL generation,CD4,+,TH,1,CD8,+,CTL,CD8,+,preCTL,17,PPT课件,Alloreactivity of T cells:CD4+,Alloreactivity of T cells,Positive,Selection,Thymus,Alloreaction,(MLR),Proliferation and Differentiation,18,PPT课件,Alloreactivity of T cellsPosit,Inflammation,lysis,ADCC,lysis,IL2,IFN,TNF,NO,2,IL2,IL4,IL5,IL2,TNF,IFN,rejection,Mechanisms of graft rejection,19,PPT课件,InflammationlysisADCClysisIL2,Tempo of rejection reaction,type of rejection,time taken,cause,chronic,months-years,unclear causes:cross reactive Ab,immune complexes,slow cellular reaction,tolerance breakdown,disease recurrence,accelerated,days,reactivation of sensitized T cells(secondary response),acute,days-weeks,primary activation of T cells,hyperacute,minutes-hours,preformed anti-donor antibodies and complement,20,PPT课件,Tempo of rejection reactiontyp,A,B&DR matching and graft survival,21,PPT课件,A,B&DR matching and graft s,Removal of T cells from marrow graft,Magnet,Magnetic antibodies,22,PPT课件,Removal of T cells from marrow,HLA and disease association,23,PPT课件,HLA and disease association23P,HLA,抗原检测法,24,PPT课件,HLA抗原检测法24PPT课件,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。,25,PPT课件,微量细胞毒试验原理25PPT课件,微量细胞毒试验,HLA I,类分型法,方法,加板：,Terasaki,板中加入特异性抗,HLA I,类分子抗体。,分离,PBMC。,制成浓度为1,10,6,/,ml,的细胞悬液。,每孔内加入1,l,细胞悬液。,室温培育30,min。,1 l,兔补体。室温培育60,min。,每孔内加入5,l 5%,伊红水溶液。室温5,min。,每孔内加37%甲醛固定。,倒置相差显微镜下观察结果。,26,PPT课件,微量细胞毒试验 HLA I类分型法方法26PPT课件,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1,h。,3,加补体后培育时间为2,h。,27,PPT课件,微量细胞毒试验 HLA II类分型法与HLA类分型法,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,PPT课件,微量细胞毒试验应用实践28PPT课件,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,PPT课件,交叉配型 用于测定受者血清中受含有抗供者HL,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。,30,PPT课件,交叉配型方法30PPT课件,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,PPT课件,细胞学分型法 细胞学分型法已被血清学和分子生,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,PPT课件,分子生物学技术为基础的HLA分型法原理32PPT课件,变异片段 变异片段 变异片段,33,PPT课件,变异片段,a b c d,ac A,TCGG,TCAAGG,CCTATC,GATTGCGTAGGC,bd ATCGGT,CAAG,GCCTATCG,ATTG,CGTAGGC,ac A,TCGG,TCAAGG,CCTATC,GATTGCGTAGGC,ad A,TCGG,TCAAGGCCTATCG,ATTG,CGTAGGC,bc ATCGGT,CAAG,G,CCTATC,GATTGCGTAGGC,34,PPT课件,a b c,根据片段组合指定等位基因,ac,ac,bc,ad,bd,ac,ef,gh,ef,ml,jk,ox,rt,wq,sv,zp,35,PPT课件,根据片段组合指定等位基因acacbcad bdacefghe,