建立阿尔茨海默症人源肠道菌群动物模型  

Establishment of a human flora-associated mouse model correlated with Alzheimer’s disease

作　　者：朱华 刘小海[2] 李卓 郭亚茜 杜晓鹏 苏磊 李永宁[2] 秦川 ZHU Hua;LIU Xiaohai;LI Zhuo;GUO Yaxi;DU Xiaopeng;SU Lei;LI Yongning;QIN Chuan(Key Laboratory of Human Disease Comparative Medicine,Institute of Laboratory Animal Science,Chinese Academy of Medical Sciences(CAMS)Comparative Medical Center,Peking Union Medical College(PUMC),Beijing 100021,China;PUMC Hospital,Academy of Medical Sciences(CAMS)&Peking Union Medical College(PUMC),Beijing 100730)

机构地区：[1]卫健委人类疾病比较医学重点实验室,中国医学科学院医学实验动物研究所,北京协和医学院比较医学中心,北京100021 [2]中国医学科学院北京协和医院,北京100730

出　　处：《中国实验动物学报》2021年第1期55-62,共8页Acta Laboratorium Animalis Scientia Sinica

基　　金：医科院重大协同创新项目(2017-12M-2-005)。

摘　　要：目的使用粪菌移植方法经口途径获得阿尔茨海默症人源肠道菌群(human flora-associated,HFA模型小鼠,并通过生物信息学、组织病理学等方法对模型进行评价。方法无菌雌性C57BL/6J小鼠28只,随机分为阿尔茨海默症模型(AD)组和对照(CON)组,分别经口途径接种阿尔茨海默症患者及健康志愿者的新鲜粪便悬液,在移植6、10周后每组安乐7只动物,取粪便进行16S r DNA检测;取脑组织和血液进行Aβ含量、细胞因子检测,脑组织进行组织病理学检查。结果CON组与AD组动物体重增长差异无显著性。α-多样性分析,在造模后两个时间点AD组Simpson指数升高(P<0.05,P<0.01),ACE(P<0.05)和Chao1(P<0.05),Shannon指数降低(P<0.05,P<0.01),与CON组比较差异有显著性。在科水平,AD组拟杆菌科丰度升高(P<0.01,P<0.05),毛螺菌科(P<0.001)、消化链球菌科(P<0.01)丰度降低;在属水平,AD组拟杆菌属丰度升高(P<0.01,P<0.05),梭菌属(P<0.01,P<0.05)、毛螺菌属(P<0.01,P<0.0001)、寄生菌属(P<0.01,P<0.001)丰度降低,与CON比较差异有显著性。β-多样性分析结果,AD组、CON组不同时间点肠型分布在相同区域,不同组别肠型分布在不同区间。2组动物的肠道菌群在微生物物种构成方面存在显著性差异(P<0.05)。AD组小鼠在建模10周时血液和脑中Aβ40含量升高(P<0.05,P<0.01),与CON组比较差异有显著性。与老年斑形成相关的细胞因子检测,IL-1β在建模6周时浓度降低(P<0.05);IL-10在建模6周时升高(P<0.01);TGF-β在建模10周时浓度降低(P<0.01),与CON组比较差异有统计学意义。组织病理学检查未见老年斑生成。结论本研究通过接种患者粪便悬液的方法建立了阿尔茨海默症人源菌群小鼠模型,模型小鼠肠道菌群的结构丰度、与老年斑生成相关细胞因子含量等指标的改变与阿尔茨海默症病人的临床表现类似。Objective To establish and evaluate a human flora-associated(HFA)mouse model from patients with Alzheimer’s disease(AD)via fecal microbiota transplantation.Methods Twenty-eight female germ-free(GF)C57 BL/6 J mice were divided into a healthy control CON group and an AD group.Six-week-old mice were orally inoculated with a 0.4 m L mixed stool suspension from 3 healthy participants CON or 3 AD patients to establish the HFA mouse model.At 6 and 10 weeks post-inoculation,fresh fecal samples were collected and examined for the V3 region of the 16 S r DNA gene.Blood sera were collected and examined for Aβand cytokine levels.Brain samples were collected,processed and stained with immunohistochemistry(IHC)for pathological examination.Results There was no difference in average body weight between the two groups.Alpha-diversity analysis showed that the Simpson’s Diversity Index(P<0.05,P<0.01)was significantly lower in the AD group than CON group.At 6 and 10 weeks post-inoculation,the abundance-based coverage estimator(P<0.05),Chao1 index(P<0.05)and Shannon function(P<0.05,P<0.01)were higher in the AD group than the CON group.At the family level at 6 and 10 weeks post-inoculation,the relative abundances of Bacteriaceae were higher(P<0.01,P<0.05),while those of Lachnospiraceae(P<0.001)and Peptostreptococcaceae(P<0.05,P<0.01)were lower in the AD group compared with the CON group.At the genus level,Bacteroides(P<0.01,P<0.05)were higher in the AD group the AD group than the CON group.The relative abundances of Clostridioides(P<0.01,P<0.05),Lachnoclostridium(P<0.01,P<0.0001)and Parasutterella(P<0.01,P<0.001)were lower in the AD group than the CON group.β-diversity analysis showed that the CON and AD groups were distributed in different quadrants,but the same groups at different stages were distributed in the same quadrants,with a significant difference between the groups(P<0.05).The Aβ40 level in serum(P<0.05)and cerebral homogenates(P<0.01)were significantly higher in the AD group than the CON group at 10 weeks post-inocu

关 键 词：阿尔茨海默症 肠道菌群 无菌小鼠 菌群人源化动物 

分 类 号：Q95-33[生物学—动物学]