血浆外泌体作为肌萎缩侧索硬化生物标志物及其功能研究

Amyotrophic lateral sclerosis（ALS） is a fatal neurodegenerative disease of unknown etiology, and biomarkers can help early diagnosis and early treatment to delay the development of the disease. However, there are still no good biomarkers. Recent studies have shown that exosomes can transmit ALS-related pathogenic proteins between cells, suggesting that exosomes may be able to transmit disease. Therefore, we hypothesized that exosomes may serve as biomarkers for ALS. Our preliminary experimental results indicated that compared with healthy controls, some plasma exosomes derived miRNAs in ALS patients have significantly change. Therefore, this project will screen for differentially expressed RNA and protein from plasma exosomes by RNA deep sequencing and proteomics on the basis of established large-scale disease cohort and skilled plasma exosome separation technology. Combined with later stage validation, we hope to screen out biomarker(s) with high specificity and sensitivity for ALS diagnosis and prognosis prediction. At the same time, we will further utilize cell and animal experiments to prove the role of exosomes in the development of ALS. This project will lay the foundation for the development of accurate minimally invasive ALS biomarkers and provide new ideas for further understanding the pathogenesis of ALS.

肌萎缩侧索硬化是一种病因不明的致死性的神经退行性疾病，生物标志物可以帮助早期诊断早期治疗以延缓疾病发展。然而目前仍无较好的疾病生物标志物。研究显示外泌体可在细胞间传播肌萎缩侧索硬化相关的致病蛋白，提示外泌体可能可以传播疾病。因此我们猜测，外泌体可以作为肌萎缩侧索硬化的生物标志物。前期预实验结果显示，同健康对照者相比，肌萎缩侧索硬化患者血浆外泌体中一些miRNA具有显著性改变。因此本项目将在已经建立的大规模疾病队列和成熟的血浆外泌体分离技术基础上，利用RNA深度测序和蛋白质组学方法，从血浆外泌体中筛选差异表达的RNA和蛋白质。结合后期验证，以最终筛选出具有一定特异性和敏感性的肌萎缩侧索硬化诊断和预后预测标志物。同时进一步结合细胞和动物实验证明外泌体在肌萎缩侧索硬化发生发展过程中的作用。本项目将为发展准确的微创的肌萎缩侧索硬化生物标志物奠定基础，并为深入认识肌萎缩侧索硬化的发病机制提供新思路。

目前肌萎缩侧索硬化症尚无有效的疾病治疗，这一现实的深层原因是对疾病的早期诊断不足和临床试验中尚无精准衡量疾病进展的有效工具。在现有文献和技术进展的支持下，我们围绕血浆外泌体的蛋白和RNA，探索其作为肌萎缩侧索硬化的生物标志物的潜力。我们的研究通过发现队列患者血浆的RNA测序和验证队列的qPCR检测，结果显示联合5种外泌体miRNA的表达水平可有效区分ALS患者和健康对照，其中miR-215-5p不仅可用于诊断ALS，而且与疾病进展速度相关。此外，外周血白细胞RNA测序和qPCR检测提示了Septin5 在ALS患者和对照之间的显著差异，进一步我们在血浆中利用神经来源的外泌体蛋白Septin5, 同样可有效的区分ALS患者和对照，并与疾病进展速度相关。我们的研究扩充了外泌体参与ALS发病机制，同时提供了未来利用外泌体miRNA和神经来源外泌体蛋白Septin5 评估ALS病情进展的新的证据。