特发性膜性肾病T细胞克隆性与抗PLA2R抗体的相关性研究

Idiopathic membranous nephropathy (IMN) is one of the most common pathological type of primary glomerular diseases. Although the pathogenesis of IMN is not clear yet, recent studies have shown autoantibodies against M-type phospholipase A2 receptor (PLA2R) could be detected in most patients with IMN but not secondary membranous nephropathy, indicating that PLA2R may be a major antigen and anti-PLA2R antibody might be a specific autoantibody in IMN. This project is aimed at investigating the correlation between T cell clonality and anti-PLA2R antibody, identifying the restricted T cell epitopes on autoantigens of PLA2R as well as the phenotype of activated T cells, and determining the nephritogenic role of anti-PLA2R autoantibody in IMN patients to evaluate the role of T-cell-dependent antigen-specific immune responses in the pathogenesis of IMN. T cell clonality will be studied using the established reverse transcriptase-polymerase chain reaction and single-strand conformation polymorphism (RT-PCR-SSCP) analysis in combination with the third complementarity determining region (CDR3) sequence analysis of the T cell receptor (TCR) Vβ genes in patients with IMN. The phenotype of activated T cells in IMN will be identified by fluorescence-activated cell sorter (FACS) cell sorting combined with RT-PCR-SSCP analysis. Anti-PLA2R antibodies will be tested in serum samples and renal tissues from patients with different pathological stages, different disease severity and patients treated with different immunosuppressives using Western Blotting and immunohistochemistry methods respectively. Correlations between anti-PLA2R antibodies and pathological stages, disease severity as well as responses to therapies in IMN patients will be subsequently analyzed. These studies may help to illustrate the limited usage of TCR Vβ genes under the stimulation of autoantigen PLA2R and demonstrate restricted T cell epitopes on autoantigen of PLA2R in IMN patients as well as the phenotype of activated T cells that might stimulate B cells to produce anti-PLA2R autoantibodies in IMN disease. Therefore, this project will be hopeful to elucidate the contribution of T-cell-dependent antigen-specific immune responses and the pathogenic role of anti-PLA2R antibody in IMN, give a new insight into the nature of autoantigen involved and provide possible specific immunoregulatory strategies against new targets in IMN patients. No domestic or overseas study in this field has been reported yet.

特发性膜性肾病(IMN)是原发性肾小球疾病的常见病理类型之一，目前发病机制不十分清楚，近年报道M型磷脂酶A2受体(PLA2R)是IMN的重要靶抗原。本项目拟应用RT-PCR-SSCP、DNA测序、流式细胞术、Western Blot等技术研究IMN患者T细胞受体(TCR)Vβ基因表达及其与抗PLA2R抗体的相关性、Vβ基因CDR3区的序列特征、活化T细胞表型与抗PLA2R抗体的相关性及抗PLA2R抗体与IMN病情的关系。来分析IMN患者T细胞克隆性与抗PLA2R抗体的相关性，阐明T细胞在自身抗原PLA2R刺激下被特异性活化，出现TCR Vβ基因的优势取用，并产生特定的T细胞活化亚型，进而辅助B细胞产生抗PLA2R抗体，导致IMN发病的免疫机制；并揭示IMN患者自身抗原PLA2R的抗原决定簇本质、活化T细胞亚型和抗PLA2R抗体的致病性，为IMN患者的特异性免疫治疗提供新途径。国内外无报道。

M型磷脂酶A2受体(PLA2R)和抗PLA2R抗体是特发性膜性肾病(IMN)重要的靶抗原和自身抗体，参与IMN发病；其他生物标志物也可能参与IMN的发生发展。本研究通过分析IMN患者T细胞克隆性与血清抗PLA2R抗体及肾组织PLA2R的相关性、分析PLA2R基因多态性、筛选IMN差异表达基因，来探讨IMN的免疫发病机制和寻找IMN新型生物标志物。结果发现，（1）抗PLA2R抗体是敏感、特异的IMN血清学标志物，抗体阳性者更易表现为肾病综合征。检测该抗体有助于老年和非老年IMN的诊断及病情监测，尤其对肾活检受限的老年患者，临床意义更大。（2）抗PLA2R抗体随治疗缓解而转阴，与疾病活动相关；动态监测抗体变化有助于评价治疗反应。（3）分析IMN患者的T细胞克隆性，发现存在TCR Vβ2、Vβ7和Vβ8基因的优势取用；肾组织TCR Vβ基因表达与血清抗PLA2R抗体之间，及其与肾组织PLA2R表达之间均存在相关性。揭示T细胞被自身抗原PLA2R活化，呈TCR Vβ基因优势取用，进而辅助B细胞产生抗PLA2R自身抗体，诱导IMN发病的免疫机制。（4）对IMN患者肾组织TCR Vβ基因CDR3区进行测序，在同一TCR Vβ基因的CDR3区，单克隆增生或寡克隆增生的序列中，均未发现不同个体之间共同的氨基酸序列；在同一个体，不同家族的TCR Vβ CDR3区，也均未发现共同使用的氨基酸序列。表明尚不能通过肾组织优势取用的TCR Vβ基因CDR3测序分析来揭示PLA2R的抗原决定簇本质。（5）PLA2R基因rs35771982位点的CC基因型与IMN发病、血清抗PLA2R抗体均具有相关性。表明CC基因型是IMN的风险因素，揭示PLA2R的基因突变导致PLA2R抗原表位暴露，刺激机体产生抗PLA2R抗体，诱发IMN的遗传学机制。（6）筛选IMN的差异表达基因，发现EIF4G1和UBL4A是IMN诊断及预后判断的两个新型重要基因标志物；核糖体相关的功能/通路受损及血小板相关的功能/通路受损促进IMN疾病进展。上述研究成果对于阐明IMN的免疫发病机制，提供IMN的新型标志物和新治疗靶点，具有重要意义。本项目还拓展了肾脏纤维化研究内容，筛选出参与肾纤维化进展的候选miRNAs：miR-21和miR-146b，探讨了肾纤维化进展的基因调控机制。目前国内外无报道。