HLA分型技术课件

,*,单击此处编辑母版标题样式,整理ppt,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,HLA,分型技术,上海交通大学医学院,彭奕冰 主任技师,1,整理ppt,Major Histocompatibility Complex(MHC),What is MHC?,HLA,H-2,Minor,histocompatibility,antigens,2,整理ppt,Significance of the MHC,role in immune response,role in organ transplantation,role in predisposition to disease,3,整理ppt,Progress in organ transplantation,4,整理ppt,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,整理ppt,Principles of transplantation,6,整理ppt,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,整理ppt,Graft versus host(GVH)disease,8,整理ppt,GVH disease in humans,9,整理ppt,The human MHC genes,10,整理ppt,The mouse MHC genes,11,整理ppt,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,整理ppt,The inheritance of MHC genes,13,整理ppt,Crossing overresults in new haplotypes,14,整理ppt,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,整理ppt,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,整理ppt,Alloreactivity of T cells:,MLR and CTL generation,CD4,+,TH,1,CD8,+,CTL,CD8,+,preCTL,17,整理ppt,Alloreactivity of T cells,Positive,Selection,Thymus,Alloreaction,(MLR),Proliferation and Differentiation,18,整理ppt,Inflammation,lysis,ADCC,lysis,IL2,IFN,TNF,NO,2,IL2,IL4,IL5,IL2,TNF,IFN,rejection,Mechanisms of graft rejection,19,整理ppt,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,整理ppt,A,B&DR matching and graft survival,21,整理ppt,Removal of T cells from marrow graft,Magnet,Magnetic antibodies,22,整理ppt,HLA and disease association,Disease,Associated alleles,Frequency in,Relative risk,patients,control,Ankylsoing spondylitis,Reiters disease,Acute anterior uveitis,Psoriasis vulgaris,Dermatitis herpetiformis,B27,B27,B27,CW6,DR3,9,79,52,87,85,9,9,9,33,26,87.4,37.0,10.4,13.3,15.4,23,整理ppt,HLA,抗原检测法,24,整理ppt,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,整理ppt,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,整理ppt,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,整理ppt,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,整理ppt,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,整理ppt,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,整理ppt,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,整理ppt,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,整理ppt,变异片段 变异片段 变异片段,33,整理ppt,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,整理ppt,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,整理ppt,