背景 阿尔茨海默病（Alzheimer’s disease, AD）属于神经退行性疾病的一种。现今研究表明，肠道菌群和AD的发生存在紧密联系。但是，现今多数研究仅局限于观察AD动物模型和AD患者的肠道菌群结构，针对肠道菌群和AD认知功能障碍及作用机制的研究并不明确。另外，目前的研究手段多采用16S rRNA基因扩增子分析肠道微生物，而培养组学技术的发展使得肠道微生物活体的提取得以实现，同时为微生物与健康、疾病之间关系的研究打下基础。所以，此项研究先借助培养组学分析SPF级APP^sew/PS1^E9双转基因AD模型（PAP）小鼠和SPF级野生型（WT）小鼠肠道微生物的组成，其次通过多组学分析研究肠道微生物与AD发生发展的相关性，进一步筛选与AD相关的微生物，同时围绕其对AD的影响展开探讨，旨在为探究AD的发病机制与潜在临床治疗手段给予新思路。

方法 1）实验组别：分组1：5月龄SPF级WT小鼠和SPF级PAP小鼠；分组2：5月龄SPF级WT小鼠、SPF级PAP小鼠、Dubosiella newyorkensis死菌灌胃的SPF级PAP小鼠和Dubosiella newyorkensis活菌灌胃的SPF级PAP小鼠；分组3：5月龄无菌WT小鼠、无菌PAP小鼠、Dubosiella newyorkensis死菌灌胃的无菌PAP小鼠和Dubosiella newyorkensis活菌灌胃的无菌PAP小鼠。

2）采集分组1小鼠的结肠内容物进行微生物的分离、培养、鉴定。

3）对分组1小鼠肠道菌群进行宏基因组测序，探讨SPF级PAP小鼠肠道菌群的组成和结构变化。

4）对分组1小鼠的粪便、血清和海马进行非靶向代谢组学检测，探讨SPF级PAP小鼠粪便、血清和海马代谢途径及代谢物的变化。

5）经由Morris水迷宫、新物体识别与Y迷宫，对分组1、2小鼠的认知功能展开评价。

6）利用病理学检测技术观察分组1、2小鼠肠道组织结构和脑组织内神经炎症。

结果 1）采集SPF级PAP小鼠和WT小鼠的粪便进行培养组学研究分析，一共分离了501株细菌，隶属于30个属和39个种，并分离获得1个新菌种M2458^T。

2）与SPF级WT小鼠相比，5月龄SPF级PAP小鼠肠道菌群的丰度和多样性低于WT小鼠。在PAP小鼠中，Libanicoccus massiliens、Paraprevoella clara和Lactobacillus amylovorus相对丰度显著增加，而Turicibacter sanguinis，Dubosiella newyorkensis, Prevotella oris, Alistipes timonensis和Neglecta timonensis相对丰度显著减少。非靶向代谢组学检测分析发现，PAP小鼠和WT小鼠相比，某些代谢途径发生显著改变，如神经递质代谢、脂质代谢、芳香族氨基酸代谢能量代谢维生素消化和吸收，以及胆汁酸代谢等。微生物-代谢相关性分析表表明，这些代谢途径的变化可能由于肠道菌群的调控，尤其是 Turicibacter sanguinis，Dubosiella newyorkensis和Prevotella oris。

3）Dubosiella newyorkensis活菌灌胃可以改善SPF级PAP小鼠肠道菌群的结构；Dubosiella newyorkensis活菌灌胃增加了磷脂酰胆碱和胆碱等参与磷脂代谢途径的代谢物含量；Dubosiella newyorkensis活菌灌胃可以改善SPF级PAP小鼠和无菌PAP小鼠的认知功能损伤；Dubosiella newyorkensis活菌灌胃可以抑制SPF级PAP和无菌PAP小鼠神经炎症的发生；Dubosiella newyorkensis活菌灌胃对PAP小鼠主要脏器无毒性作用。

结论 1）培养组学通过分离出活的细菌，为后续实验建立菌库，提供物质基础。

2）PAP小鼠肠道微生物结构和组成发生改变，可能调控宿主的代谢途径。

3）Dubosiella newyorkensis活菌可能通过调节肠道菌群和调节甘油磷脂代谢紊乱影响AD发生发展。

Background Alzheimer's disease (AD) is a neurodegenerative disease. At present, studies have found that gut microbiota is closely related to the occurrence and development of AD. However, most of the studies are limited to the observation of gut microbiota structure in AD patients and AD animal models, while investigations on the relationship and mechanism between the disturbance of gut microbiota and AD cognitive function are not clear. In addition, most of the current research methods use 16S rRNA to analyze the composition and structure of gut microbiota, while the development of culturomics technology can obtain live intestinal microorganisms, and lay a material foundation for the study of the causal relationship between microorganisms and health and disease. Therefore, this topic first uses culturomics to study the composition of gut microbiota in specific pathogen free (SPF) APP^sew/PS1^E9 double transgenic AD model (PAP) mice and SPF wild type (WT) mice, then studies the correlation between gut microbiota and the occurrence and development of AD through multiomics analysis, further screens AD related probiotics, and discusses their impact on AD, in order to provide new ideas for the study of AD pathogenesis and potential clinical treatment methods.

Methods 1) Grouping 1 included SPF WT mice and SPF PAP mice at 5 months old, group 2 included SPF WT mice, SPF PAP mice, SPF PAP mice gavaged with dead Dubosella newyorkensis (SPF DDN )and SPF PAP mice gavaged with live Dubosella newyorkensis (SPF LDN), group 3 included GF WT mice, GF PAP mice, GF PAP mice gavaged with dead Dubosella newyorkensis (GF DDN) and GF PAP mice gavaged with live Dubosella newyorkensis (GF LDN).

2) Colonic contents of mice in group 1 were collected for isolation, culture and identification of microbiota.

3) The gut microbiota of group 1 mice were determined by metagenomics to investigate the composition and structural of gut microbiota of SPF PAP mice.

4) The feces, serum and hippocampus of group 1 mice were detected by non targeted metabolomics to explore the changes of metabolic pathways and metabolites in the feces, serum and hippocampus of SPF PAP mice.

5) Morris water maze, Y maze and new object recognition were used to evaluate the cognitive function of mice in group 1 and 2.

6) Pathological examination was used to observe intestinal tissue structure and neuroinflammation in brain tissue of mice in groups 1 and 2.

Results 1) The feces of SPF PAP mice and WT mice were collected for culturomics analysis. A total of 501 strains of microbiota belonging to 31 genera and 40 species were isolated, and a new strain M2458^T was obtained.

2) Compared with SPF WT mice, SPF PAP mice at 5 months of age had lower abundance and diversity of gut microbiota than WT mice. In PAP mice, the relative abundance of  Libanicoccus massiliens, Paraprevoella clara and Lactobacillus amylovorus increased significantly, while the relative abundance of  Turicibacter sanguinis, Dubosiella newyorkensis, Prevotella oris, Alistipes timonensis and Neglecta timonensis decreased significantly. Non targeted metabolomic analysis showed that compared with WT mice, PAP mice had significant changes in some metabolic pathways, such as neurotransmitter metabolism, lipid metabolism, aromatic amino acid metabolism, energy metabolism, vitamin digestion and absorption, and bile acid metabolism. The microbial metabolism correlation analysis showed that the changes of these metabolic pathways may be due to the regulation of gut microbiota, especially Turicibacter sanguinis, Dubosiella newyorkensis and Prevotella oris.

3) The structure of gut microbiota in SPF PAP mice could be improved by gavage of live Dubosiella newyorkensis, live Dubosella newyorkensis increased the content of metabolites involved in the glycerolphospholipid metabolism pathway such as phosphatidylcholine and choline, live Dubosella newyorkensis can improve the cognitive impairment of SPF PAP mice and GF PAP mice; live Dubosella newyorkensis could inhibit the occurrence of neuroinflammation in SPF PAP and GF PAP mice; The main organs of PAP mice were not toxic to Dubosiella newyorkensis by gavage.

Conclusion 1) By isolating live bacteria, culturomics provides a material basis for the establishment of a bacterial library for subsequent experiments.

2) The abnormal of gut microbiota structure and composition in PAP mice may regulate the metabolic pathway of the host.

3) Dubosella newyorkensis may influence the occurrence and development of AD by regulating gut microbiota and glycerolphospholipid metabolism disorder.