一种罕见的肉芽肿性脑膜-脑-脊髓炎的特异抗体-靶抗原的鉴定、功能及致病机制研究

Autoimmune-inflammatory diseases in central nervous system (CNS) represent a broad spectrum, including monophasic, multiphasic, and progressive disorders and ranging from highly localized forms to multifocal or diffuse variants. Human common CNS autoimmune-inflammatory diseases, including multiple sclerosis and neuromyelitis optica disorder spectrum have been well known as autoantibody-independent astrocytopathy. However, another rare astrocytopathy with specific antibody, targeting to glial fibrillary acidic protein (GFAP), are still poorly understood due to limited studies. Since June 2013, we have encountered some rare cases with a pattern-specific astrocyte-antibody in CSF and/or serum. This antibody is targeting to the glial fibrillary acidic protein (GFAP). The clinical and pathological data were certified that such cases were highly similar to granulomatous meningoencephalomyelitis (GME) in animals. Loss of GFAP in astrocyte, apoptosis and autophahgy in astrocyte were found in our previous study. Animal study showed a pathogenic role of GFAP antibody to the astrocyte. A scientific hypothesis was raised in our present project that astrocyte injury and inflammatory response in GME is associated with signal pathway regulated by GFAP antigen and its antibody. GFAP is critical to pathogenesis of GME. Therefore, study will be performed in the present project, including: (1) clinical value of GFAP will be elevated; (2) we will clarify that GFAP is the sole antigen to astrocyte-antibody by gene knockout technology;(3) a critical role to pathogenesis of GFAP will be certified by cell and animal model; (4)based on our previous result, we will explore astrocyte apoptosis and autophagy in Akt signal pathway, which is controlled by GFAP antigen- GFAP antibody response. Our project is helpful for clinical and basic study of GME in the future.

申请者在前期发现了一组以星形细胞抗体为标记的罕见疾病，GFAP可能是该抗体的靶抗原。临床与病理学证实它与犬的肉芽肿性脑膜脑脊髓炎高度相似。星形细胞GFAP的脱失、凋亡和自噬现象亦被证实。动物实验证实GFAP抗体具有损伤星形细胞的效应。我们提出假设：GFAP在疾病中起关键作用，GFAP抗体-抗原轴调控下游相关信号通路、介导星形细胞损伤与炎症发生发展。因此，本项目拟开展研究:(1)建立基于GFAP表达的分子预测模型，评价这一指标的临床适用价值；(2)敲除鼠GFAP基因,证实星形细胞抗体的唯一靶点是GFAP；(3)建立基于GFAP抗体诱导的细胞与动物模型，通过控制GFAP表达对模型炎症反应的影响来阐明GFAP在疾病发生、进展中的关键作用；(4)根据前期工作的基础，阐明在GFAP抗体作用下，GFAP是否通过Akt信号通路调控、影响星形细胞凋亡与自噬。本项目对该疾病的临床与基础研究均具有重要意义。

自身免疫性胶质纤维酸性蛋白星形细胞病（glial fibrillary acidic protein astrocytopathy，GFAP-A)、也称自身免疫性GFAP脑膜脑脊髓炎，是一组累及脑膜、脑和脊髓为主的新谱系疾病。然而GFAP-A的病因和发病机制不详。本团队在GFAP-A的研究处于国际前列，率先报道了中国GFAP-A病例并世界范围首次描述了系列GFAP-A的病理学特征，证实了脑部大量星形细胞增殖、损伤伴随小胶质细胞的活化。基于临床表现、辅助检查、病理学特征和治疗预后研究，我们证实该组病例酷似发生于动物犬的肉芽肿性脑膜-脑-脊髓炎。初步的研究亦证实星形细胞抗体的靶抗原有GFAP-α和/或ε。而且证实，来源于患者的IgG抗体具有致星形细胞损伤的效应, 其致病机制涉及星形细胞的凋亡与自噬过程。靶标分子GFAP在疾病中起关键的作用，GFAP通过介导下游的凋亡等相关信号通路产生星形细胞的损伤效应。项目研究完成下列内容：（1） 通过基因敲除后检测的方法，阐明抗星形细胞抗体是特异靶向 GFAP 蛋白：项目通过敲除C57小鼠的GFAP基因，TBA鉴定证实星形细胞的抗体是靶向GFAP蛋白。（2） 确认 GFAP 在本疾病谱中起关键作用，与星形细胞的损伤直接相关；项目通过临床样本的研究，从病理学和生物标志物检测证实GFAP蛋白在该疾病中起关键作用，跟星形细胞损伤直接相关。细胞模型和动物模型同样证实了GFAP的关键作用。（3） 探讨 GFAP 调节下游炎症反应信号通路与效应分子，阐述具体作用分子机制：项目通过临床样本的研究，从生物标志物检测证实了GFAP下游的炎症信号通路改变。细胞模型和动物模型探讨了相关的分子机制。以上研究在组织、细胞、动物三个层次展开，并通过分子机制研究进行纵向的突破，目前资料已经总结，共发表论文13篇，获得实用新型专利一项，申报专利1项和培养6名硕士，在站博士后2名，多次参加全国学术会议并做报告推广研究结果。而研究的推动及所获得的研究成果可帮助我们深入认识这种新的谱系疾病，对于制定合理的治疗方案，采取有针对性的干预措施，减轻患者神经功能残障至关重要，并为相关靶点应用于临床转化医学奠定理论基础。