自身抗体1G3调控DDX5对血脑屏障通透性的影响及机制研究

The disruption of blood-brain barrier plays an important role in the pathogenesis of central nervous system autoimmune disease. Although neuronal cytotoxic effect of some autoantibodies have been demonstrated, the effect of autoantibody on blood-brain barrier is still unclear. We have obtained a brain-targeting monoclonal autoantibody 1G3, and our preliminary data indicate that 1G3 could increase the cleaved caspase-3 level and inhibit the viability of glial cells; 1G3 specifically target to DDX5 and significantly decreased the DDX5 level in nuclear of glial cells. Previous study shows that the decease of DDX5 in nuclear can inhibit the accumulation of β-catenin into cell nuclear, and induce apoptosis and decrease of the tight junction protein expression through blocking the β-catenin/TCF pathway. Therefore we hypothesized that 1G3 could mediate blood-brain barrier disruption by regulating DDX5 level and the downstream β-catenin/TCF pathway. By using multiple molecular biology tools, cell culture, and mice model, we comprehensively investigate the effect and mechanism of autoantibody 1G3 on blood-brain barrier permeability. Together, this study will provide not only a novel theoretical basis on the pathogenesis of central nervous system autoimmune disease, but also provide new molecular targets for the diagnosis and targeted therapy of central nervous system autoimmune disease.

血脑屏障损伤在中枢神经系统自身免疫性疾病的致病机制中占有重要地位。尽管某些自身抗体的神经毒性作用已被证实，但是自身抗体对血脑屏障的影响仍然未知。我们已获得靶向脑组织的全人源单克隆自身抗体1G3，前期研究发现1G3可上调神经胶质细胞内活化的caspase-3表达并降低细胞活力；1G3可靶向DDX5，并显著减少细胞核内DDX5的含量。有研究表明，细胞核内DDX5的减少会抑制β-catenin在核内集聚，从而阻断β-catenin/TCF通路，促进细胞凋亡并抑制紧密连接蛋白的表达。因此我们推测1G3可能通过调控DDX5及其下游β-catenin/TCF通路介导血脑屏障损伤。本课题拟在分子水平、细胞水平和动物水平综合分析自身抗体1G3对血脑屏障通透性的影响及其机制，为阐明中枢神经系统自身免疫性疾病的发病机制提供实验依据，并为此类疾病的特异性诊断和靶向治疗提供新的分子靶点。

血脑屏障（blood brain barrier, BBB）损伤在中枢神经系统自身免疫性疾病的致病机制中占有重要地位。尽管某些自身抗体的神经毒性作用已被证实，但是自身抗体对BBB的影响仍然未知。我们已获得靶向脑组织的全人源单克隆自身抗体1G3，前期研究发现1G3可上调神经胶质细胞内活化的caspase-3表达并降低细胞活力；1G3可靶向DDX5，并显著减少细胞核内DDX5的含量。有研究表明，细胞核内DDX5的减少会抑制β-catenin在核内集聚，从而阻断β-catenin/TCF通路，促进细胞凋亡并抑制紧密连接蛋白的表达。本研究从分子水平、细胞水平和动物水平综合分析自身抗体1G3对BBB通透性的影响及其机制。Biacore生物大分子互作分析结果显示1G3与其靶蛋白DDX5呈现高亲和力结合、快解离的特点。体外实验研究发现，1G3对BBB细胞活力没有显著影响，且1G3对BBB没有促进细胞凋亡的作用。1G3能够显著降低BBB细胞HA和HMEC细胞中DDX5的表达水平和BBB紧密连接蛋白的表达水平。体内实验研究证实，1G3能够显著降低小鼠BBB紧密连接蛋白的表达，且1G3处理后的小鼠组织器官没有明显病理改变。本研究能够为阐明中枢神经系统自身免疫性疾病的发病机制提供实验依据，并为此类疾病的特异性诊断和靶向治疗提供新的分子靶点和理论依据。