阿尔茨海默病患者肠道菌群结构及菌群代谢产物的神经毒性研究

Alzheimer's disease (AD) is the aging population-specific neurodegenerative disease and has lead to big problem in economic and social fields. Now, the pathogenesis of AD is not clear and we still have no effective clinical treatment. The hypothesis of AD might be related to alteration of intestinal microbiota and increased toxic metabolites, which could cause inflammation and nerve injury, but there is still no direct evidence. The small sample of our experiment results showed that blood endotoxin (microbial metabolites, can cause inflammation) level in AD patients was significantly higher than that of in normal control group, and the number of gut neurotoxin-produced cyanobacteria also increased significantly in AD patients. This study intends to detailed analyse intestinal microbiota in AD patients by using high-throughput sequencing and detect systemic inflammatory status in AD patients. At the same time, the fecal extracts containing the bacterial metabolites was prepared, and the neurotoxicity of the bacterial metabolites of AD patients was studied by the method of co-culture of the fecal extracts and the nerve cells. And the intestinal barrier model in vitro was used to detect whether bacteria metabolites of AD can damage the intestinal mucosal barrier and lead to toxic substances into the blood circulation.The project design specific test to validate scientific hypotheses, and to explore possible role of intestinal microbiota and its metabolites in the occurrence and development of AD. The results of this project will provide a theoretical basis for prevention and treatment of AD by regulation of intestinal microbiota.

阿尔茨海默病(AD)是人口老龄化特有的神经退行疾病，已引起不可忽视的经济与社会问题。目前AD的发病机理未明，尚无特效防治方法。已有假说提出AD可能与肠道菌群失调，毒性代谢产物增多，继而引起机体炎症以及神经损伤相关，但目前仍无直接证据。本项目小样本预实验结果发现AD患者的血内毒素（微生物代谢产物，能引起机体炎症）水平明显高于正常对照组，同时肠道内产神经毒素的蓝细菌数量明显增加。本研究拟采用高通量测序对AD患者肠道菌群进行详尽解析，并测定AD患者机体炎症状态。同时制备含有菌群代谢物的粪便提取液，利用提取液与神经细胞共培养的方法研究AD患者菌群代谢物的神经毒性。并采用体外肠屏障模型研究AD患者菌群代谢物能否损伤肠屏障，导致毒性物质进入血循环。本项目设计具体试验验证科学假说，并探讨肠道菌群及其代谢物在AD发生发展中的可能作用，研究结果为将来通过调节肠道菌群对AD进行预防及治疗提供理论基础。

AD是人口老龄化特有的神经退行疾病，目前尚无特效防治方法。本研究AD患者体内炎症水平、菌群代谢物的神经毒性与对肠屏障损伤与对照组无统计学差别。而高通量测序结果表明AD患者肠道菌群组成与正常老年对照存在显著差异。AD肠内厚壁菌门与变形菌门丰度相对升高，而拟杆菌门丰度相对下降,厚壁菌门与拟杆菌门的比例增高在AD的高危疾病肥胖人群中已得到广泛证实，重度抑郁症患者肠内变形菌门含量也显著增加。大肠埃希菌/志贺菌在AD组数量明显增高,这类菌可产生LPS，我们的结果表明外周血LBP的含量也明显增高,与产LPS的细菌数量增加相互验证，积累的LPS能够促炎并增强Aβ的形成，可引起神经炎症及促进神经退行性变。Blautia在AD患者肠道内增高，已有研究发现其在AD前期疾病轻度认知障碍患者肠道中已有增加。可产生神经毒素的蓝细菌（Cyanobacteria）在AD组中数量明显增加。而罗斯氏菌(Roseburia)在AD患者中数量减低，该菌是丁酸盐产生菌，对肠屏障与血脑屏障通透性具有保护作用。我们实验结果中一些有害肠道细菌数量增高以及一些益生菌的数量下降，可能与AD的发生、发展相关。我国进行临床第三阶段AD治疗的药物GV-971作用机制之一为抑制肠道菌群失调及相关的菌群代谢产物积聚。如果肠道菌群失调与AD发生发展中的作用得到证实，未来可通过重塑肠道菌群对AD进行预防与治疗。. 另外，本研究根据AD与正常对照差异肠道菌群建立AD筛选模型，其敏感性为83.33%，分类错误率为25%。目前国际AD诊断标准中PET成本高，腰穿为侵入性方法，不被老年人普遍接受。肠道菌群的检测费用相对低，且是非损伤性方法，未来特定肠道菌群检测有望成为AD的辅助诊断方法。本项目研究结果为AD的筛查与防治提供理论基础。今后可采用全基因组测序寻找相关代谢通路，分离差异菌群，进行菌群灌注等实验，对差异细菌在AD中的作用进行深入研究。