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HLA 分型技术

HLA 分型技术. 上海交通大学医学院 彭奕冰 主任技师. Major Histocompatibility Complex (MHC). What is MHC? HLA H-2 Minor histocompatibility antigens. Significance of the MHC. role in immune response role in organ transplantation role in predisposition to disease. Genetic barriers to transplantation.

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HLA 分型技术

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  1. HLA分型技术 上海交通大学医学院 彭奕冰 主任技师

  2. Major Histocompatibility Complex(MHC) • What is MHC? • HLA • H-2 • Minor histocompatibility antigens

  3. Significance of the MHC • role in immune response • role in organ transplantation • role in predisposition to disease

  4. 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. Principles of transplantation

  6. 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. Graft versus host (GVH) disease

  8. GVH disease in humans

  9. The human MHC genes

  10. The mouse MHC genes

  11. 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. The inheritance of MHC genes

  13. Crossing overresults in new haplotypes

  14. MHC products expressed on cells If Jack and Jill have four children; Bo, Kim, Mo and Lee They’ll 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. Differential expression of MHC antigens • 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.)

  16. CD4+TH1 CD8+preCTL CD8+CTL Alloreactivity of T cells: MLR and CTL generation

  17. Alloreactivity of T cells Alloreaction (MLR) Thymus Positive Selection Proliferation and Differentiation

  18. TNF, NO2 IL2, TNF, IFN  IL2, IL4, IL5 lysis IL2, IFN  Mechanisms of graft rejection Inflammation ADCC lysis rejection

  19. type of rejection cause time taken minutes- hours preformed anti-donor antibodies and complement hyperacute reactivation of sensitized T cells (secondary response) days accelerated primary activation of T cells acute days-weeks unclear causes: cross reactive Ab, immune complexes, slow cellular reaction, tolerance breakdown, disease recurrence months-years chronic Tempo of rejection reaction

  20. A, B & DR matching and graft survival

  21. Magnet Removal of T cells from marrow graft Magnetic antibodies

  22. HLA and disease association

  23. HLA抗原检测法

  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。 　　　Ｔ淋巴细胞只表达HLA I分子，如欲测定HLA II类分子，需用Ｂ细胞。

  25. 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106/ml的细胞悬液。 每孔内加入1l细胞悬液。 室温培育30min。 1 l兔补体。室温培育60min。 每孔内加入5l 5%伊红水溶液。室温5min。 每孔内加37%甲醛固定。 倒置相差显微镜下观察结果。

  26. 微量细胞毒试验 HLA II类分型法 与HLAＩ类分型法区别 1．须用富含Ｂ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。

  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，法医鉴定等。

  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。

  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。

  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。

  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. 变异片段 变异片段 变异片段

  33. a b c d ac ATCGGTCAAGGCCTATCGATTGCGTAGGC bd ATCGGTCAAGGCCTATCGATTGCGTAGGC ac ATCGGTCAAGGCCTATCGATTGCGTAGGC ad ATCGGTCAAGGCCTATCGATTGCGTAGGC bc ATCGGTCAAGGCCTATCGATTGCGTAGGC

  34. 根据片段组合指定等位基因 ac ef ac ox gh rt ac ef wq bc ad ml sv jk zp bd

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