人体血管内皮细胞新移植抗原基因鉴定及其多态性研究

The waiting for kidney transplantation in our country currently is counted by millions and millions. Among these, over a third or more of them could be sensitized against the polymorphic endothelial allo-antigens. The HLA antigens are the major targets of the immune response against the donor tissue. However, rejection may occur in the absence of antibodies against the HLA antigens of the donor. It has therefore been proposed that other antigens expressed in the graft endothelium, and not normally detectable in peripheral blood lymphocytes, may be additional targets that can cause allograft loss. We and others have used endothelial cells isolated from umbilical veins (HUVEC) to detect antigens that may be responsible for kidney allograft rejection in patients in whom antibodies against HLA cannot be detected even using the most sensitive techniques. These polymorphic endothelial antigens could be a major source of sensitization and a cause of unexplained graft loss in patients with negative HLA crossmatches. Understanding these antigens could have an important impact that could also benefit recipients of other organs such as heart and lung. Presence of such antibodies, in the complete absence of antibodies against HLA, was strongly associated with decreased survival of kidney allografts obtained from deceased donors. We now would like to use immunological and protein identification techniques to recognize and characterize these antigens. Experiments to be performed include: 1). Antibodies to human umbilical cord vein endothelial cells (HUVEC) determined by flow cytometry; 2). Identification of sera with antibodies against polymorphic endothelial antigens. 3). Immunoprecipitation of endothelial cell antigens. 4). Protein identification by mass spectrometry. 5). Once the genes are identified, nucleotide sequencing will be used to map the polymorphisms and to correlate them with the serologic results. 6). The next step will be to produce recombinant antigens representing the polymorphic immunogenic sequences identified, which can be used for the development of an immunoassay based on Luminex bead multiplex technology. Our group has documented experience and published experiments using all of these approaches in connection with the study of MICA antigens and antibodies and their role in organ transplantation. The techniques of single-antigen Luminex bead assays for antibodies against polymorphic antigens are fully developed and operational.Methods, procedures and materials for the various components are ready for deployment immediately when support becomes available. Preliminary evidence suggests that characterization of these endothelial antigens could have an important effect on the success rate of organ transplantation, which would impact the health of many patients with organ failure for whom transplantation is the best treatment option. Our group is in a unque position to solve this problem now.

HLA作为主要组织相容性抗原在移植免疫起着重要作用，但患者在缺乏抗供者HLA抗体和交叉配型阴性的情况下仍然发生对移植物的排斥反应。前期研究结果提示内皮细胞(EC)特异性抗体由于不被T和B淋巴细胞的交叉配型试验检出，临床上HLA交叉配型反应阴性的肾移植出现不明原因的排斥反应很可能与该EC抗原有关。探索EC抗原的编码基因是当前急需解决的关键问题。我们应用HUVEC细胞模型和细胞流式技术对EC抗体血清样本进行了鉴定，并从血清中提纯该抗体，再通过免疫沉淀直接捕获相应的EC抗原。利用蛋白质组学和质谱分析技术，确定该EC抗原的人体编码基因。随即开展对该基因测序分型，血清学相关性分析和人群该EC基因多态性调查。制备常见等位基因型的重组蛋白质，制成多抗原液态芯片Allo-抗体检测系统。确定新EC移植抗原和检测其抗体特异性为建立器官移植的内皮细胞虚拟配型提供实用的实验数据，有利于预防临床器官移植免疫排斥反应

器官移植排斥反应仍然是移植器官存活的重要障碍，与移植排斥相关的移植抗原除ABO血型抗原，HLA和MICA多态性抗原外，还有一些Non-HLA抗原。研究表明这些抗原都与血管内皮细胞抗体（AECAs）有关，并在器官移植排斥中发挥作用，但病人血清中存在的Non-HLA 抗体确切作用于何种内皮细胞（EC）表面抗原尚未完全清楚。本研究应用对肾排斥病人血清AECA抗体特异性分析，用从人脐静脉血清分离的HUEVC作为靶细胞对AECA阳性血清进行吸附和洗脱，实现了对HLA和MICA抗原捕获，成功建立了用血清抗体对靶抗原的免疫沉淀方法。应用建立的抗体分离，免疫沉淀和质谱技术我们发现了一种新的内皮细胞移植抗原角蛋白1（KRT1）可能与肾移植排斥反应相关。同时我们用抗KRT1单克隆抗体检测到人脐静脉血管内皮细胞膜表面有KRT1的表达，通过基因克隆技术将人群中常见的三种KRT1多态性蛋白进行了表达和重组蛋白的分离纯化。用这种重组蛋白设计ELISA实验对肾移植病人的血清抗KRT1特异性抗体进行检测，结果显示：肾移植随访病例血清KRT1抗体阳性率为20.8%(53/255)。对肾移植后病例的血清肌酐（Cr）浓度进行分析，发现在Cr>120umol/L的病例组有29.9%（23/77）的受者被检测出存在KRT1抗体，而在Cr≤120umol/L的肾移植病例中的KRT1阳性率为16.8%(30/178)，提示KRT1抗体阳性与肾移植后肾功能的慢性损伤存在统计学上的相关性(P<0.05)。进一步对高滴度KRT1抗体的特异性和相应病例的KRT1基因型进行分析，我们发现KRT1抗体属于一种抗KRT1的自身抗体。随着对临床上大样本器官移植病例回顾性调查分析，我们将揭示KRT1作为血管内皮细胞移植抗原在器官移植体液排斥中发挥作用，这为设计新的实验方法，评估器官移植风险和指导临床预防排斥用药具有重要的临床意义。