散发型包涵体肌炎中p62/SQSTM1诱导炎性反应的分子机制

Sporadic inclusion body myositis (sIBM) is a rare disease, but is the most common slowly progressive inflammatory muscle disease among people aged over 50 years old. The pathogenesis of sIBM is still unclear, and it has been hotly debated between inflammation and degeneration pathways. No effective treatment is available so far. We previously found that p62 protein is the most common protein aggregate (91.3%) observed in muscle biopsy of Chinese sIBM patients. P62 is an important functional protein involved in autophagy and immunomodulation. Rare variants in the p62/SQSTM1 gene were suggested to contribute to the genetic susceptibility of sIBM by using whole-exome sequencing. In addition, the patients with p62/SQSTM1 rare variants showed significantly higher expression of MHC-I and MHC-II genes by using whole-genome expression analysis and RT-PCR. Therefore, we hypothesise that the impairment in p62-related autophagy induces the inflammatory reaction in sIBM. In this project, a cell model transfected with p62/SQSTM1 cDNA and an animal model with p62 aggregate in skeletal muscle will be generated. Changes of autophagy, its relationship with inflammatory molecules, and pathology of muscle will be observed by immunohistochemistry and electron microscopy. Expression level of autophagic and inflammatory molecules will be analysed by using RT-PCR, western blotting, and co-immunoprecipitation. We aim to confirm that the activation of inflammasomes and up-regulation of MHC are associated with high expression of p62, and to further identify the mechanism of p62/SQSTM1 induced inflammation. This study will improve our understanding of pathogenesis of sIBM.

散发型包涵体肌炎（sIBM）是中老年人最常见的炎性肌肉疾病，其致病机制的研究长期徘徊在炎症和变性病之间。我们前期发现自噬功能蛋白p62是中国sIBM肌肉病理中阳性率最高的蛋白沉积标记物（91.3%），全外显子测序显示p62/SQSTM1基因变异为sIBM的遗传易感因子，携带该基因变异的患者MHC-I及MHC-II基因有显著高表达。因此p62相关的自噬异常可能诱导了sIBM的炎性反应。本研究拟采取转染质粒诱导产生高表达p62的细胞模型，采取注射重组腺病毒载体诱导骨骼肌p62高表达的大鼠模型，采用免疫组织化学染色和电镜技术观察细胞自噬与炎性因子的关系和骨骼肌病理改变，通过RT-PCR、蛋白印迹法和免疫共沉淀测定自噬和免疫炎性因子的表达和含量。通过上述研究预期证实炎性通路中的炎性小体激活、MHC上调和p62升高有关，明确p62诱导免疫炎性反应的分子机制，为探讨sIBM致病机制提供理论据。

散发型包涵体肌炎（sIBM）是中老年人最常见的慢性进行性炎性肌肉疾病，在我国相对罕见，其致病机制尚不清楚。欧洲学者在sIBM患者的血清中发现了抗cN1A抗体，但我国患者尚未进行该抗体检测。本项目成功构建了高表达p62蛋白细胞模型，并对细胞在不同时期凋亡情况及炎症因子的表达进行检测，未发现有明显细胞凋亡情况，炎性因子MHC-II的表达亦无明显的差别。这提示p62蛋白可能为自噬通路异常的中间产物，并非上游启动因子。本项目还建立了国内首个已知多中心sIBM登记队列，结果将我国诊断标准起病年龄下限调整至40岁可能减少漏诊率。联合检测p62、SMI-31和TDP43蛋白沉积有助于提高诊断率。相比于欧洲患者，国内患者屈髋无力的出现率较高。本项目对19例sIBM患者抗cN1A抗体进行了检测，其敏感度和特异度分别为42.1%和97.8%。抗体阳性组比阴性组的平均起病年龄更大。41.2%患者出现肌纤维内cN1A蛋白异常沉积，偶与p62蛋白共定位沉积。该抗体对诊断sIBM具有重要价值。对20例sIBM或疑似sIBM及33例管聚集改变的患者进行全外显子测序，在一例先证者中发现SBF基因的一个纯合移码突变，且存在家系共分离，功能学验证该突变导致编码蛋白表达下降。发现11/33人（33%）带有已知致病或可能致病的罕见变异。STIM1基因中c.764A>T (p.Glu255Val)和SCN4A基因中 c.1333G>C (p.Val445Leu)为新发位点。对ALG14基因中的p.Ala11Thr变异位点进行功能学验证，提示编码蛋白有显著下降，有致病可能。本项目为未来制定我国sIBM诊疗指南有很重要的意义，为进一步研究发病机制提供了新的思路。