免疫细胞亚群在肠道微生物介导帕金森病发病中的作用和机制研究

Parkinson's disease (PD) is a neurodegenerative disease characterized by abnormal aggregation of alpha-synuclein (α-syn) in the cytoplasm of dopaminergic neurons in the substantia nigra. The changes in enteric microorganism can affect the homeostasis of the immune system and participate in the pathogenesis of PD through multiple links. Our team found that PD patients had abnormal gut microorganisms, and many their immune related genes expressions in peripheral blood were obviously up-regulated, including the signal pathway of T cells and TNF receptor related factor-3 gene which was related to the activation of microglia induced by α-syn. Compared to the normal mice, the immune related gene expressions in peripheral blood and different brain regions of PD model mice changed significantly. AEP and PIKE-L activity associated with abnormal aggregation or scavenging of α-syn were also affected by immune inflammation. Given the heterogeneity of different immune cell subsets, this study plans to use 16S rRNA sequencing and droplet single-cell sequencing to analyze subtle features of intestinal microorganisms and single leukocyte expression profiles in peripheral blood of PD patients and normal controls. All experiments design are based on the previous results. Our purpose is to screen the key immune cell subsets that respond to the changes of intestinal microorganisms abundance in the progress of PD, to elucidate the mechanism of the specific immune cell subsets in the pathogenesis of PD, and to clarify the interaction pathway among the intestinal microorganisms, immune cells and nervous system.

帕金森病（PD）是以黑质多巴胺能神经元胞浆内α-突触核蛋白（α-syn）异常聚集为特征的神经退行性疾病。肠道微生物改变可影响机体免疫系统的稳态，并通过多个环节参与PD发病进程。本团队前期发现：PD患者存在肠道菌群异常，且外周血中T细胞等免疫相关信号通路上调、与α-syn诱导小胶质细胞活化有关的TNF受体相关因子-3基因表达明显上调；PD模型小鼠外周血及各脑区的免疫相关基因表达水平显著改变；与α-syn异常聚集或清除相关的AEP及PIKE-L活性均受免疫炎症影响。鉴于不同免疫细胞亚群功能的不均一性，本研究拟在前期结果的基础上，采用16s rRNA测序和液滴单细胞测序技术分析PD病人与正常对照的肠道微生物及外周血单个核细胞表达谱的精细特征差异，筛选PD进程中响应肠道微生物丰度变化的关键免疫细胞亚群，明确特定免疫细胞亚群在PD发病进程中的作用机制，阐明肠道微生物-免疫细胞-神经系统相互作用途径。

帕金森病（PD）是以黑质多巴胺能神经元胞浆内α-突触核蛋白（α-syn）异常聚集为特征的神经退行性疾病。肠道微生物改变可影响机体免疫系统的稳态，并通过多个环节参与PD发病进程。前期研究提示肠道微生物可能通过外周免疫途径参与PD发生，鉴于不同免疫细胞亚群功能的不均一性，本研究首先通过流式细胞术分析了PD病人的外周血T淋巴细胞亚群，发现PD病人的初始型CD8+ T细胞显著减少、老年型CD4+ T细胞显著增多，并在此基础上构建了个体化PD风险预测模型。其次，本研究采用液滴单细胞测序技术分析了PD病人与正常对照的外周血单个核细胞表达谱的精细转录组特征差异，根据参考标记基因共定义了29种免疫细胞亚群，发现与健康对照相比，PD病人外周血中的经典型单核细胞显著增多，其中PD病人高表达的差异基因主要富集在NF−kappa B signaling pathway、IL−17 signaling pathway等免疫相关通路，进一步通过WGCNA及蛋白互作网络分析等，发现了经典型单核细胞中的Fos和CTSS基因可能在PD中发挥关键作用。同时，本研究采用16s rRNA测序技术明确了PD病人的粪便微生物组成特征，通过与PD病人的临床特征进行关联分析，发现了Butyricicoccus及Christensenellaceae_R-7_group两种菌在粪便微生物中的比例变化对PD的发生具有重要意义。多组学联合分析提示，Butyricicoccus菌和Christensenellaceae_R-7_group菌与经典型单核细胞的囊泡转运及免疫调节功能密切相关。进一步通过粪菌移植的小鼠实验，证明了PD病人的粪便微生物具有激活免疫细胞、促进多巴胺能神经元死亡的能力。本研究阐明了肠道微生物-免疫细胞-神经系统相互作用途径，为寻找PD治疗的新靶点提供了理论依据。