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朱华,刘小海,李卓,郭亚茜,杜晓鹏,苏磊,李永宁,秦川.建立阿尔茨海默症人源肠道菌群动物模型[J].中国实验动物学报,2021,29(1):55~62.
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
投稿时间:2020-06-02  
DOI:10. 3969 / j.issn.1005-4847. 2021. 01. 008
中文关键词:  阿尔茨海默症  肠道菌群  无菌小鼠  菌群人源化动物
英文关键词:Alzheimer’s disease  intestinal flora  germ-free mice  human flora-associated animal
基金项目:
作者单位E-mail
朱华 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 zhuh@ cnilas.org 
刘小海 中国医学科学院 北京协和医院,北京 100730  
李卓 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  
郭亚茜 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  
杜晓鹏 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  
苏磊 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  
李永宁 中国医学科学院 北京协和医院,北京 100730 liyongning@ pumc.org 
秦川 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 qinc@ cnilas.org 
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中文摘要:
       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 rDNA 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。
英文摘要:
       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) C57BL/ 6J mice were divided into a healthy control CON group and an AD group. Six-week-old mice were orally inoculated with a 0. 4 mL 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 16S rDNA gene. Blood sera were collected and examined for Aβ and 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-inoculation. In the AD group, the interleukin (IL) - 1β level was lower at 6 weeks ( P< 0. 05), the IL-10 level was higher at 10 weeks ( P< 0. 01), and the transforming growth factor-β level was lower at 6 weeks (P< 0. 01) post-inoculation post-inoculation compared with the CON group. Pathological examination using IHC staining of the brain showed no senile plaques. Conclusions An HFA mouse model derived from AD patients was established via fecal microbiota transplantation. The main advantage of using bacteria from AD patients was that the GF mice were well colonized.
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