免疫相关性全血细胞减少症患者骨髓造血细胞膜靶抗原TRAPPC4的研究

Immune-related pancytopenia (IRP) was first found by our center which characterized by pancytopenia caused by bone marrow hematopoietic cell autoantibody-mediated bone marrow destruction or suppression. After more than 10 years research, the immune mechanism of IRP has been gradually revealed, but there are still many mechanisms remain unclear. Exploring and identifying its own antibody against target antigen is particularly important for further study of pathogenesis, diagnosis and targeted treatment. Our research group previously screened the auto-antigens by SEREX method (serological analysis of recombinant cDNA expression libraries). We established the recombinant cDNA expression libraries derived from K562 cells and performed immunoblotting analysis of this library with sera from IRP patients and normal healthy controls. The results showed that TRAPPC4 (trafficking protein particle complex subunit 4) might be act as a target antigen for IRP bone marrow hematopoietic cells. Based on the previous studies, we are going to use ELI-spot to screen and locate the antigen epitopes of TRAPPC4, and compare it with some known antigens (for example, EPOR and lacroferrin) and certain microbial pathogenic antigen epitopes. Meanwhile, we will use flow cytometry, RT-PCR, Western blot and some other methods to verify its antigenicity in vivo and in vitro, including the whole process of DC cell antigen presentation, activation of antigen-specific Th lymghocyte, activation of B lymghocyte, formation of specific antibodies and the attacking target cells, hoping to clarify the underlying causes of IRP pathogenesis. Further more, we will explore the construction of IRP mouse model by antigen peptide, and create the conditions for further study of the pathogenesis of IRP.

免疫相关性全血细胞减少症（IRP）是我中心首次发现的一类由于机体产生抗骨髓造血细胞自身抗体而抑制和（或）破坏骨髓、引起外周血细胞减少的自身免疫性疾病。寻找IRP自身抗体针对的靶抗原对进一步揭示疾病发病机制尤为重要。课题组前期采用重组cDNA表达文库的血清学分析法（SEREX）初步筛选出TRAPPC4可能为靶抗原。本课题拟在前期研究的基础上，应用酶联免疫斑点试验（ELI-spot）、流式细胞术、RT-PCR、Western、细胞培养等方法，定位TRAPPC4的抗原表位，比较是否与一些致病的抗原表位相重叠；从体内、体外及动物模型验证其DC细胞抗原呈递、激活Th细胞、活化B细胞、生成特异性抗体、攻击靶细胞的过程，从而验证其抗原性，为进一步阐明IRP免疫发病的根本病因奠定基础。同时探索抗原肽诱导构建IRP小鼠模型，为深入研究IRP发病机制创造条件。

IRP发病的免疫机制十分复杂，尽管前期研究取得了一些进展，使我们对该疾病的免疫发病机制有了初步认识，但仍有许多问题亟待解决，首当其冲的便是寻找和验证IRP发病的根源——靶抗原。.前期我们通过构建了K562细胞的噬菌体展示文库，运用SEREX法成功筛选出7种可能为IRP患者骨髓造血细胞的靶抗原蛋白，TRAPPC4（trafficking protein particle complex subunit 4）即为其一。本课题继续对IRP发病的上游激活因素进行探讨和验证，应用噬菌体随机肽库进行亲和筛选，结合ELISA挑取噬菌体阳性克隆测序，与TRAPPC氨基酸序列比对，并联合T细胞表位预测软件，定位了TRAPPC4的抗原表位；并用ELISAspot方法证实该肽段可以激活TH2细胞；MHC-肽四聚体流式细胞术检测到初治的HLA-DRB1 15:01的IRP患者中存在一群抗原特异性CD4+ T细胞。ELISA检测到初治IRP患者血清中针对TRAPPC蛋白的抗体滴度升高。发现IRP初治患者骨髓CD34+细胞表面TRAPPC4的表达升高，而CD235a+的有核红细胞表面及CD15+的骨髓粒细胞表面TRAPPC4的表达无升高，推测CD34+的骨髓造血干祖细胞可能是IRP自身抗体攻击的靶细胞。另外EBV感染可影响B淋巴细胞的免疫功能，刺激B淋巴细胞的增殖和转化，导致自身免疫疾病的发生。为探讨EB病毒在IRP患者免疫发病机制中发挥的作用，我们对IRP患者的EBV感染和B淋巴细胞的功能抗原进行了检测。结果表明，IRP组EBVCA-IgM阳性率和CD19+细胞EBV-DNA拷贝阳性率均显著高于正常对照组(p<0.05)，近期感染EB病毒IRP患者CD21、CD23、CD5、CD80、CD86在CD19+B细胞表面的表达率明显高于近期未感染EB病毒IRP患者(p<0.05)，并且近期感染EB病毒IRP患者预后明显差于近期未感染EB病毒IRP患者，与EBVCA IgM阴性的IRP患者相比，具有抗 EBVCA IgM阳性的IRP患者获得缓解所需的时间明显更长。提示EBV感染可能是激活CD19+B淋巴细胞并进一步损害骨髓造血的主要因素。.本研究的研究成果展示了TRAPPC激活Th细胞、活化B细胞、生成特异性抗体、攻击靶细胞的过程，从而验证其抗原性，为进一步阐明IRP免疫发病的根本病因奠定基础，为临