KIR基因家族测序分型技术及HLA-C—KIR精细组合型对肝移植急性排斥影响的系列研究

The highly polymorphic killer cell immunoglobulin-like receptors (KIRs) gene cluster play a important role in tranplant immunoity, tumor immunoity and the resistance of infection. However, the simultaneously sequencing-based typing (SBT) assay for KIR frame genes and the commerical KIRs SBT kit were not available in the worldwide by now. The present widely used PCR-SSP assay for identification the presence or absence of KIR frame genes can not be used in the determination of high-resolution KIR gene profile. Based on the published documents, the outcomes of HLA-C-KIR polymorphisms associated with acute rejection (AR) in liver transplantation differed remarkably due to the multiple sources of donor-recipient pair and also different ethnic background of the study group. The techniques and reagents used in HLA and KIR genotyping also differed. In order to explore a novel approach to promote long-term graft and patient survival by modulation of HLA-C and KIR interactions, the adult orthotopic liver transplantation (OLT) receipts of southern Hans survived more than one year after transplantation were included in this study, all were transplanted at a single liver transplantation center. According to the indication of liver transplantion, these receipts were divided into two groups: termed primary carcinoma of the liver and the end-stage liver disease. This study aimed to develop the sequencing-based typing (SBT) assay for all fourteen functional KIR frame genes and investigate the potential susceptible and protective factors for the AR in liver transplantation by SBT for HLA-C locus and KIR family genes. We compare the the frequecies of "donor HLA-C and receipt KIR compound profile" of AR group with the non-AR group and also the unrelated healthy control with an aim to establish the permissive and non-permissive HLA-C-KIR compound profiles, these profiles confirmed by the subsequent NK-cell cytotoxicity assay, will be able to provide valuable information in the personalized immunosuppressive therapy for future liver transplantation.

具有高度多态性的KIR基因家簇，在移植免疫、肿瘤免疫和机体抗感染中均发挥重要作用，但迄今国际上尚无KIR同步测序分型技术及商品化测序试剂盒，不能鉴定精细的KIR等位基因组合型。在HLA-C、KIR与肝移植急性排斥的关联研究中，由于病例资料来源、种族背景、移植适应症类型以及HLA、KIR分型方法的不同，得到的结果存在较大的差异并难以重复。本项目研究和建立全部的14个功能性KIR框架基因的测序分型技术，采用前期工作中已获专利授权的HLA测序技术，以源于同一移植中心、供/受者均为南方汉族、肝移植后成活1年以上的良性终末期肝病和恶性原发性肝癌的成人原位肝移植病例为研究对象，在高分辨水平的基础上探讨KIR、HLA-C与肝移植急性排斥的易感/保护性影响因素及作用机制；通过分组对照比较以及NK细胞杀伤功能实验，建立"供者HLA-C-受者KIR基因组合型"精细相容谱，为开展个性化免疫抑制治疗提供理论依据。

杀伤细胞免疫球蛋白样受体（KIRs）与HLA I 类分子配位，在移植免疫中发挥重要作用，但迄今国际上仍无商品化KIR测序分型试剂。.本项目在国内外首次建立了适合于高通量化、产业化的全部14个功能性KIR基因同步测序分型的方法，已获中国发明专利授权（专利号：201710284545.3）并申请了美国发明专利（申请号：15696120）。每个KIR基因的全部编码区序列分别采用3~5对特异性PCR引物进行扩增，共设计PCR引物56对、测序引物230条。14个KIR基因可在同一PCR扩增条件下进行同步扩增、同步测序。. 以所建立的KIR测序分型方法，辅以创建的“组特异性PCR扩增再测序”法鉴定模棱两可的结果、分子克隆测序鉴定可能的KIR新等位基因，完成了南方汉族无关个体（n=306）14个KIR基因测序分型，共获得44500余条序列，检出116种等位基因，其中46种为WHO 正式命名的新等位基因，占当前国际上已公布的KIR等位基因总数的5.49%（46/838），有助于提高中国人群KIR基因分型的准确性；检出KIR基因精细组合型总计258种，其中KIR AA、AB、BB型分别为124、119、15种；系统获得了精细水平KIR基因家族多态性的基础数据。.本项目研究“供者HLA-C—受者KIR”与肝移植急性排斥（AR）的相关性，基于单一肝移植中心来源的143例供-受者对血样，发现：（1）供者携带HLA-C1C1基因型，AR组中受者发生急性排斥的比例为47.1%，显著低于无急性排斥组（75.4% ；P<0.05)，为保护性因子；（2）供者携带HLA-C1C2基因型、“受者携带2DS4且供者携带HLA-C2”、“受者携带2DS4*00101且供者携带HLA-C2”，AR组中受者出现急性排斥的比例分别为52.9%、50.0%、43.8%，均显著高于无急性排斥组（P<0.05），提示：接受HLA-C1C2基因型供肝的移植患者移植后需加强监控，受者携带2DS4而供者携带HLA-C2的基因组合型应尽可能避免。研究还发现受者中共检出74种KIR等位基因，精细KIR基因组合型对AR无显著影响。携带常见型KIR基因组合型的2DS4+ NK细胞，对HLA-C1/C2的白血病原始幼稚细胞的杀伤率高于HLA-C1/C1基因型的幼稚细胞。本项目已完成全部研究内容，培养研究生4人，发表论文31篇。