B细胞调节的免疫反应在脑海绵状血管畸形（CCM）中的机制性研究

The cerebral cavernous malformation (CCM) is a common cerebrovascular anomaly. Cerebral hemorrhage caused by CCM can lead to serious complication, and even death. The molecular mechanisms involved in the pathophysiology of CCM await characterization. There is no known medicine treatment to alter the course of this disease. The applicant has performed the long-run and deep study on the CCM diseases, including the infiltration of immunocompetent cells, immune complex and complement deposits, and T-B aggregates in CCM lesions. This phenomenon is associated with vascular endothelial permeability. In our data, we found B cell depletion therapy could reduce CCM lesional progression and hemorrhage. However, the definitive mechanism of B cell-mediated immune response in the CCM disease is unclear. We hypothesize that immune complex and antibody-dependent complement activation on the endothelia, increasing the vascular endothelial proliferation and permeability via the activation of cellular multiple signaling pathways, promoting CCM lesional progression and hemorrhage. This proposal will investigate 1. In our recently developed Pdcd10+/-Trp53-/- mice, the investigation of T- cells being readily available to provide help for B- cells via the inhibition of T-B aggregates. 2. Which complement pathway(s) involved in the lesional progression and hemorrhage of CCM diseases. 3. In vitro and in vivo study, the investigation of the mechanistic role of the immune complex in the promotion of vascular endothelial proliferation and permeability, via the activation of multiple signaling pathways. With a fresh perspective, this study would provide proof of principle of its mechanistic implications of B cell mediated immune response in the CCM disease. It lays a theoretical foundation of immunotherapy in this disease and provides a new target for treating this disease. This project has a certain significance of transformation.

脑海绵状血管畸形（CCM）是脑血管畸形的一种，由其导致的脑出血可致严重的并发症甚至死亡。CCM发病机制尚不明确，尚无有效的药物治疗。申请人对CCM进行长期深入的研究发现，CCM中有免疫细胞、免疫复合体和补体存在、和T-B细胞聚集等，这些与内皮细胞(EC)渗透性紧密相关。B细胞耗竭减少了CCM病变进展和脑出血，但机制不详。我们假设免疫复合体和抗体依赖的补体激活沉积在EC上, 通过细胞内多个信号通路的活化, 增加了EC的增殖和渗透性, 促使CCM病变的进展和出血。本课题拟研究 1、在转基因鼠中阻断T-B细胞聚集，阐明B细胞发挥作用是否由T细胞辅助；2、哪种补体激活途径参与CCM病变进展、出血；3、体内外实验探讨免疫复合体激活多信号转导通路，使EC繁殖和渗透性增加的机制。本研究从新的角度，试图探讨B细胞调节的免疫反应在CCM中的作用机制，为CCM治疗提供新靶点和理论依据，有一定转化意义。

脑海绵状血管畸形(CCM)可以引起脑出血、癫痫甚至死亡。目前疾病进展和出血机制不祥，且无有效的药物治疗。我们的前期研究发现了CCM病变内有大量的免疫细胞聚集，并且证实B细胞在CCM病变原位克隆扩增。在此，我们通过激光捕获显微切割分离患者病变内的成熟B细胞，通过测序其抗体的可变区序列制成了重组单克隆抗体，利用免疫染色、免疫沉淀、质谱分析等实验技术明确了重组抗体识别细胞骨架蛋白为自发抗原,免疫染色显示重组单克隆抗体与补体在病变内共沉积。因此，CCM病变内细胞骨架蛋白作为自发性抗原促使B细胞介导的免疫反应引起了组织损伤，这些结果促使我们进一步理解该疾病的发展，能够为今后的研究提供新的方向及为今后的治疗提供依据。.随后我们利用内皮细胞中特异性敲除Ccm1基因后构建的CCM小鼠模型，通过对小鼠小脑神经血管单元进行RNA-seq分析发现CCM病变小鼠的细胞中Fn1基因表达量显著增加，Fn1基因编码的fibronectin参与细胞连接及转移过程。进一步的研究显示周细胞表达的fibronectin是CCM病变的重要来源，并且周细胞fibronectin的表达受到KLF4与pSMAD3共同调控。使用fibronectin的抑制剂RGDS peptide可使小脑和视网膜的CCM病变缩小。最后，通过检测临床手术样本我们还发现人CCM病变同样有丰富的fibronectin沉积以及pSMAD3和KLF4阳性周细胞，Fibronectin的干预可抑制CCM病变的发展，因此，fibronectin有可能作为CCM疾病治疗的药物靶标。