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抗N-甲基-D-天冬氨酸受体脑炎的B 细胞免疫组库研究
Alternative TitleStudy of B Cell Immune Repertoire in Patients with Anti-Nmethyl- D-aspartate Receptor Encephalitis
冯京京
Contributor王晶
2021-06
Abstract抗N- 甲基-D- 天冬氨酸受体脑炎( anti-N-methyl-D-aspartate receptor encephalitis , anti-NMDARE ) 是一种由抗体和B 细胞表面受体( B cell receptor, BCR)介导的、通过影响自身NMDA 受体进而造成中枢神经系统神经损害的自身免疫性疾病。这些抗体和BCR 的作用靶点为NMDA 受体的NR1亚基,因此,脑内以NR1 抗原为特异性靶点的抗体或BCR 是该病分子机制研究的重点对象之一。B 细胞免疫组库(Immune repertoire, IR)是一种通过测序获得BCR 和抗体基因序列来研究B 细胞介导的自身免疫病疾病特征及发病机制的重要方法。目前已有三篇文献研究了国外人群抗NMDAR 脑炎患者的NR1 阳性BCR 序列,他们找到NR1 阳性BCR/抗体的方法效率较低、找到的NR1 阳性BCR/抗体种类较少,亟须寻找更高效的研究方法。此外,他们的研究未从免疫组库角度为抗NMDAR 脑炎患者与健康人群之间的差别提供更多线索。另一方面,流行病学及组织学结果提示畸胎瘤作为该脑炎的诱因之一,但目前尚无进一步的分子生物学层面证据证实二者之间的关联,对比分析畸胎瘤患者与抗NMDAR 脑炎患者的B 细胞IR 特征,可对二者之间的关系进行基因层面的探讨。有鉴于此,本论文开展了以下三个研究,从B 细胞IR 角度对疾病特征进行了探讨。 第一,建立了NR1 阳性BCR 基因捕获方法与NR1 阳性BCR 数据集。本研究通过流式细胞术分选了患者脑脊液(cerebral spinal fluid,CSF)中能与NR1抗原结合的B 细胞,并利用单细胞BCR/抗体基因扩增和测序方法对NR1 阳性BCR 基因序列进行了捕获,共成功获取来自12 例抗NMDAR 脑炎患者CSF 的逾100 个NR1 阳性B 细胞的BCR 序列,经过序列比对最终获得了44 种重链和38 种轻链。与上述三篇已发表文献比,本研究采用的NR1 阳性BCR 基因捕获方法效率更高,同时找到了更多NR1 阳性BCR 序列。 第二,利用生物信息学方法探讨了患者的NR1 阳性BCR 基因数据集的整体特征。本研究发现脑炎患者间有公共克隆,NR1 阳性BCR 具有明显的V/J 基因使用偏好性。此外,利用公共数据库中其他中枢神经系统自身免疫病和健康人的B 细胞免疫组库测序数据,验证了所获得的NR1 阳性基因数据集的特异性,为该数据集作为抗NMDAR 脑炎潜在标志物奠定了基础。 第三,通过传统免疫组库高通量测序和生物信息学方法对比分析了另7 例抗NMDAR 脑炎患者、4 例畸胎瘤患者(无脑炎)和4 位健康人外周血B 细胞免疫组库特征。本研究发现:两组疾病组的外周血B 细胞免疫组库多样性低于健康组;三组在V/J 基因使用偏好性、突变率等方面具有明显差异;部分脑炎患者和畸胎瘤患者具有相对较高的B 细胞免疫组库相似性,结合流式细胞术结果显示的脑炎组和畸胎瘤组均存在NR1 阳性B 细胞这一结果,可以推断两组患者的B 细胞免疫组库相似性可能与NR1 抗原有关。本研究既分析了抗NMDAR脑炎患者与健康人在外周血B 细胞免疫组库方面的差异,也为后续进一步揭示畸胎瘤作为该脑炎的可能诱因提供了线索。 通过研究一和研究二的结果,我们得出以下结论:不同的抗NMDAR 脑炎患者脑内存在相同抗原表位,患者脑内存在明显的寡克隆增生现象;通过研究三的结果,我们得出以下结论:健康人与抗NMDAR 脑炎患者在外周血B 细胞免疫组库特征方面具有差异;血清呈抗NMDAR 抗体阴性的畸胎瘤患者体内可以存在NR1 抗原暴露现象。
Other AbstractAnti-N-methyl-D-aspartate receptor encephalitis (anti-NMDARE) is mainly mediated by antibodies and B cell receptor (BCR) specifically binding to the NR1 subunit of NMDA receptor in the central nervous system, these antibodies cause neurological damage to the central nervous system by affecting the NMDA receptors. Therefore, the antibody or BCR specifically targeting NR1 antigen in brain is one of the key objects in the molecular mechanism study of this disease. Immune repertoire (IR) is an important method to study the characteristics and pathogenesis of B-cellmediated autoimmune diseases by obtaining BCR or antibody gene sequences through sequencing. At present, there are only three reports of NR1-positive BCR from foreign patients with anti-NMDARE. They only found a small number of NR1-positive BCR/antibody genes and their methods were less efficient, so it is urgent to find a more efficient method for the study of NR1-positive B cell IR. In addition, their study did not provide additional clues about the differences between patients with anti-NMDARE and healthy people from the perspective of IR. On the other hand, epidemiology and histological results have shown that teratoma is one of the triggers of this encephalitis, but there is no further molecular biology evidence of the correlation between teratoma and anti-NMDARE. Comparing B cell IR characteristics of patients with teratoma and patients with anti-NMDARE can explore the relationship between them at the level of gene. Therefore, we carried out the following three aspects of research to explore the characteristics of these diseases from the perspective of B cell IR. First of all, we established a NR1-positive BCR gene capture method and NR1-positive BCR gene data set. We selected B cells in cerebral spinal fluid (CSF) that could bind to NR1 antigen by flow cytometry, and then we captured their BCR genes by single cell IR amplification and sequencing. Finally, we got gene sequences of more than 100 NR1-positive B cells from 12 patients with anti-NMDARE, and after sequence alignment, we finally obtained 44 kinds of heavy chains and 38 kinds of light chains. Compared with the three published literature mentioned above, the NR1-positive BCR gene capture method we adopted is more efficient, and we also found more NR1-positive BCR sequences. Secondly, we explored the overall characteristics of patients' NR1-positive BCR gene data set by bioinformatic methods. We found that there were common clones among patients with anti-NMDARE, and these NR1-positive BCR showed obvious V/J gene usage preference. In addition, the specificity of the obtained NR1-positive BCR gene data set was verified by using BCR data of other immune-mediated disorders of the central system and healthy people from the public databases, which laid a foundation for the data set to be used as potential markers of anti-NMDARE. Thirdly, we analyzed the B cell IR characteristics in the peripheral blood of the other 7 patients with anti-NMDARE, 4 patients with teratoma (without anti-NMDARE) and 4 healthy people using traditional immune repertoire high-throughput sequencing and bioinformatic methods. We found that the diversity of B cell IR in peripheral blood of these two disease groups were lower than that of the healthy group; there were significant differences in the preference of V/J gene usage, mutation rate and so on among these three groups; some patients with anti-NMDARE and some patients with teratoma had relative high similarity in B cell IR. Combined with the results of flow cytometry that NR1-positive B cells were found in both the teratoma group and the encephalitis group, we deduced that the similarity of B cell IR between these two groups may be related to NR1 antigen. Our study not only reflected the differences in peripheral blood B cell IR between patients with anti-NMDARE and healthy people, but also provided clues for further study on teratoma as a trigger of this encephalitis. Based on the results of the first study and second study in this paper, we reached the following conclusions: different patients with anti-NMDARE had the same epitope, and there was obvious oligonoclonal hyperplasia in their brain; through the results of the third study, we reached the following conclusions: there were differences between healthy people and patients with anti-NMDARE in the characteristics of peripheral blood B cell immune repertoire; patients only with teratoma whose anti-NMDAR antibodies test were negative in serum can have exposed NR1 antigen in their bodies.
Keyword抗N-甲基-D-天冬氨酸受体脑炎 免疫组库 NR1 阳性BCR 传统免疫组库高通量测序 中国人群
Subtype博士
Language中文
Degree Name理学博士
Degree Discipline健康心理学
Degree Grantor中国科学院心理研究所
Place of Conferral中国科学院心理研究所
Document Type学位论文
Identifierhttp://ir.psych.ac.cn/handle/311026/39606
Collection健康与遗传心理学研究室
Recommended Citation
GB/T 7714
冯京京. 抗N-甲基-D-天冬氨酸受体脑炎的B 细胞免疫组库研究[D]. 中国科学院心理研究所. 中国科学院心理研究所,2021.
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