HLA分型技術(shù)PPT課件

1、1Major Histocompatibility Complex(MHC)What is MHC?HLAH-2Minor histocompatibility antigens第1頁/共35頁2Significance of the MHC role in immune response role in organ transplantation role in predisposition to disease第2頁/共35頁3Progress in organ transplantation第3頁/共35頁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頁/共35頁5Principles of transpl

3、antation第5頁/共35頁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頁/共35頁7Graft versus host (GVH) disease第7頁/共35頁8GVH disease in

4、humans第8頁/共35頁9The human MHC genesclass IIclass IDPDQDRBCABCADPDQDR122211931 221 3第9頁/共35頁10The mouse MHC genesIDKAEclass IIclass Iclass Iclass III(C4, factor-B,TNF, etc.)第10頁/共35頁11Polymorphism of MHC antigens (based on phenotype) HLA基因系統(tǒng)的多態(tài)性及一些主要基因座位的等位基因數(shù)正式命名顯示多態(tài)性的HLA基因座位有31個，共2 320個等位基因。 圖中所示為常見

5、基因座位的等位基因數(shù)。第11頁/共35頁12The inheritance of MHC genesJackJillDPDQDRBCA1234BoKimMoLee13412342第12頁/共35頁13Crossing overresults in new haplotypes第13頁/共35頁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頁/共35頁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頁/共35頁16Alloreactivity of T cells:MLR and CTL generationCD4+TH1CD8+CTLCD8+preCTL第16頁/共35頁17Alloreactivity of T cellsT CELLT CELLDRW11DRW17PositiveSelectionThymusAlloreaction(MLR)Proliferation and Differentiation第17頁/共35頁18InflammationlysisADCClysisIL2, IFN TNF, NO2IL2, IL4, I

8、L5IL2, TNF, IFN rejectionMechanisms of graft rejection第18頁/共35頁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頁/共3

9、5頁20A, B & DR matching and graft survival第20頁/共35頁21Removal of T cells from marrow graft MagnetMagnetic antibodies第21頁/共35頁22HLA and disease association第22頁/共35頁23HLA抗原檢測法第23頁/共35頁24微量細胞毒試驗原理當特異性抗體與淋巴細胞表面HLA分子結(jié)合，激活補體，使細胞溶解。在顯微鏡下可見細胞被活性染料著色。淋巴細胞只表達HLA I分子，如欲測定HLA II類分子，需用細胞。第24頁/共35頁25微量細胞毒試驗 HLA

10、 I類分型法方法1.加板：Terasaki板中加入特異性抗HLA I類分子抗體。2.分離PBMC。制成濃度為1 106/ml的細胞懸液。3.每孔內(nèi)加入1 l細胞懸液。4.室溫培育30min。5.1 l兔補體。室溫培育60min。6.每孔內(nèi)加入5 l 5%伊紅水溶液。室溫5min。7.每孔內(nèi)加37%甲醛固定。8.倒置相差顯微鏡下觀察結(jié)果。第25頁/共35頁26微量細胞毒試驗 HLA II類分型法與HLA類分型法區(qū)別1須用富含細胞的淋巴細胞懸液。2抗體與細胞培育時間為1h。3加補體后培育時間為2h。第26頁/共35頁27微量細胞毒試驗應用實踐 個體HLA分型可用于移植前供者和受者配型，親子鑒定，法

11、醫(yī)鑒定等。第27頁/共35頁28交叉配型 用于測定受者血清中受含有抗供者HLA的抗體。如受者血清中具有抗供者紅細胞血型抗原抗體和/或抗白細胞抗原抗體，會引起超急排異反應。所以移植前應作紅細胞血型測定，以及測定患者血清中是否具有抗供者HLA抗體。第28頁/共35頁29交叉配型方法1. 分離供體PBMC。2. 分離受者血清，作1：2，1：4和1：8稀釋。加入Terasaki板，每孔1 l。其余步驟同微量細胞毒試驗。如結(jié)果為陽性，表明受者血清中具有抗供者HLA抗體。第29頁/共35頁30細胞學分型法 細胞學分型法已被血清學和分子生物學方法代替。單相混合淋巴細胞培養(yǎng)實驗還在使用。第30頁/共35頁31分子生物學技術(shù)為基礎的HLA分型法原理HLA的多態(tài)性是由基因中核苷酸序列突變引起的。同一