PSYCH OpenIR  > 健康与遗传心理学研究室
自闭症患者肠道菌群的结构变化及NS乳酸菌干预机制研究
其他题名Structural Changes of Gut Microbiota in children with Autistic Spectrum Disorders and the intervention mechanism of specific Lactobacillus NS strains
吴晓丽
学位类型博士
导师金锋
2018-06
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业认知神经科学
关键词自闭症(孤独症) 肠脑 肠漏 微生物一肠一脑轴 益生菌
摘要

研究背景:
    自闭症是一种严重的神经发育障碍,它对社会和患者家庭造成极其严重的经济和医疗负担,并且目前临床中尚未有有效的治疗方法。据保守估计我国自闭症患病率为1 070,并且呈逐年上升趋势。自闭症患者不仅表现出行为症状,还伴随着诸多典型的生物学症状,如免疫功能异常、胃肠功能紊乱、多种过敏等问题。尽管人们多年来致力于寻找致病基因,但其患病率急剧上升的现状完全不符合群体遗传学的哈迪一温伯格平衡,表明外在因素对其发生的影响远大于遗传因素。事实上,肠道微生物在自闭症的发生与发展中起重要作用,自闭症与个体的肠道微生物失衡及肠一脑轴异常密切相关。由于婴幼儿的肠脑发育与大脑发育几乎同步,因而在发育关键期,任何影响其肠道微生物的因素均可增加自闭症风险。
    肠道微生物可通过其代谢产物、免疫、神经内分泌、以及迷走神经等途径影响大脑和行为。目前以肠道微生物为靶点干预自闭症正在成为研究热点,主要方式包括饮食干预、药物干预、粪菌移植和益生菌干预,其中益生菌以其有效性和安全性得到较多认可。本研究在关注近年来与肠道微生物相关的自闭症研究基础上,尝试对自闭症患者进行特定益生菌干预,从而为了解共生微生物在自闭症发病中的角色和相应对策展开深层次的认识。
研究目的:
1.建立健康儿童以及自闭症儿童肠道微生物数据库,分析自闭症儿童肠道微生物的构成,尝试通过调整和改变肠道微生物组份来干预自闭症。
2.通过自闭症儿童肠道微生物与对照儿童肠道微生物结构的比较研究,确定自闭症儿童肠道微生物的变化,确定自闭症相关微生物标记物。
3.通过检测微生物一肠一脑轴功能相关生理生化指标变化,分析自闭症儿童与健康对照儿童生理生化指标差异,探讨肠道微生物影响自闭症的分子途径。确定与疾病密切相关的生理指标及其变化,为临床诊断和干预提供参考。
4.通过分析直接监护人填写的评估问卷,对比自闭症儿童的行为异常和消化道症状在特定乳酸菌干预前后的变化,评估患者行为认知症状和胃肠道症状的改善程度。
5.研究NS乳酸菌(包括瑞士乳杆菌NS8菌株和发酵乳杆菌NS9菌株)对于自
闭症干预的有效性及其作用机理。
研究材料与方法:
1.收集自闭症儿童的问卷量表、粪便样本和血液样本。
2.使用自闭症治疗评估表(Autism Treatment Evaluation Checklist,  ATEC)评估患者的自闭症症状。
3.使用胃肠道症状严重程度指数问卷(Gastrointestinal Severity Index,  GSI)评估患者的胃肠道症状。
4.对粪便样本进行16s rRNA和ITS 1高通量测序分析肠道微生物组成,研究自闭症儿童肠道菌群结构及其与健康对照儿童的差异,分析NS乳酸菌干预对肠道微生物的影响。
5.比较自闭症儿童与正常发育儿童血液中与微生物一肠一脑轴相关的免疫指标、神经递质和激素等指标,探究差异指标以及经过NS乳酸菌干预后的变化情况,揭示NS乳酸菌干预对自闭症的有效性以及作用机理。
6.比较自闭症儿童(未干预)、接受NS乳酸菌干预的自闭症儿童与正常发育儿童组间的菌群及其代谢差异、血液指标和行为认知指标的差异,以及这些指标间的联系,探究肠道微生物影响自闭症的机制。
研究结果:
1.自闭症儿童不仅表现出明显的行为认知症状,还伴随显著的胃肠道症状,存在肠道微生物失衡(包括细菌组和真菌组)和生理生化异常(血清补体C3含量降低,Y_氨基丁酸(GABA)和谷氨酸(Glu)含量升高)。
2.  NS乳酸菌干预显著改善自闭症患者的胃肠道症状和自闭症症状。干预三个月后,自闭症儿童的胃肠道症状明显改善(总GSI分数降低),具体表现在:粪便性状改善、粪便气味改善、胀气改善、不明原因发怒行为减少、夜醒次数减少。NS乳酸菌干预还明显减轻了患者的自闭症严重程度(总ATEC得分减少,各个分量表分数降低),具体表现在:干预三个月后,自闭症儿童语言交流增加,社交情况改善,感知觉症状减轻,身体健康水平提高。
3.  NS乳酸菌干预明显改善自闭症儿童的肠道微生物失衡状态。
4.  NS乳酸菌干预明显改善自闭症儿童的生理生化异常。干预三个月后,自闭症儿童血清补体C3含量显著增加,GABA含量显著降低。
5.  NS乳酸菌干预对生理和行为的改善与对菌群的调整相关,比如补体C3含量与Romboutsia和Terrisporobacter丰度呈负相关,而GABA含量与Romboutsia和Terrisporobacter丰度呈正相关。
研究结论:
    综上,自闭症儿童肠道细菌和真菌与健康对照儿童相比存在显著差异。异常的肠道微生物可通过微生物一肠一脑轴中免疫途径和神经内分泌途径影响自闭症;NS乳酸菌干预能够调整自闭症患者的肠道微生物,使之趋近于正常对照儿童,并通过免疫途径和神经内分泌途径改善自闭症。
创新点:
    本研究首次对所谓“不治之症”的自闭症人群实施了NS乳酸菌组合干预。获得干预前后的行为、生理、生化和第二基因组变化,并进行了定性定量的综合评估,获得翔实丰富的数据。首次在分子水平上证实NS乳杆菌对于自闭症患者的肠道微生物以及微生态的影响,改善了患者体内免疫水平和神经内分泌功能,对自闭症的心理行为症状和生理生化水平有显著提升。

其他摘要

Research Background:
    Autism spectrum disorders (ASD) is a severe neurodevelopmental disorder. ASD has brought a heavy economy and health burden for social and families. So far clinical level does not have effective treatment for the disorder. According to conservative estimation, the prevalence of ASD in China is about 1%,and keeps increasing year by year. The ASD patients not only show behavioral symptoms, but also have many typical biological symptoms which were omitted by clinical doctors for long time, such as abnormal immune function, gastrointestinal disorders and multiple allergies.Within three decades, people have been tracing the disease genes, but the sharp increase of the disorder does not correspond with the population genetic law of Hardy-Weinberg  Equilibrium,  that  means  the  disorder  is  more  influenced  by environmental factors instead of genes. In fact, gut bacteria play more important role in neural system development and occurrence of ASD which is known to be closely associated to the unbalanced of gut microorganism and the abnormality of gut- brain axis.  Since  the  brain  development  accompanied  with  gut-brain  development simultaneously, any factor that affect their gut microbes could increase the risk of ASD during the critical period of individual development.
    It is well known that gut microbes affect the brain and behavior through their metabolites, immune, neuroendocrine, and vagus pathway. Therapies with the gut bacteria as a target intervention for ASD is becoming a new hotspot, which includes dietary  intervention,  drug  intervention,  fecal  microbiome  transplantation,  and probiotics intervention. Among the applications, probiotics is more concerned with its effectiveness and safety.
The present study aims to understand the interaction of the gut bacteria and ASD, and supplies two special commensal probiotic strains on the patients with ASD, to compare the biological and psychological change of the patients.
Research objects:
1 .Establish gut microbiota database of Chinese healthy children and ASD children,analyze the gut microbiota composition of ASD children, and try to treat ASD through gut microbiota regulation.
2. Compare the gut microbiota of healthy children and that of ASD children, identify the gut microbiota abnormalities of ASD children, and find reliable ASD related bacteria biomarkers.
3. Detect the changes of biochemical indicators related to microbiota-gut-brain axis,analyze the differences of biochemical indicators between healthy and ASD children,explore the effects of gut microbiota in ASD, identify the biochemical indicators closely related with ASD, and hope to provide reference for the clinical diagnosis and intervention of ASD.
4. Analyze the questionnaires filled out by the guardians of ASD children, compare the changes of behavioral and gastrointestinal symptoms before and after NS lactobacillus intervention, and evaluate the improvements after intervention.
5. Study the effectiveness and possibly mechanism of NS lactobacillus intervention which include Lactobacillus helveticus NS8 and lactobacillus fermentium NS9.
Materials and methods:
1 .Collect the questionnaires, stool samples and blood samples of ASD children.
2. Evaluate the behavioral and cognitive symptoms of ASD children using the Autism Treatment Evaluation Checklist (ATEC).
3. Evaluate the gastrointestinal symptoms of ASD children using the Gastrointestinal Severity Index (GSI).
4. Analyze the microbiota of stool samples using 16s rRNA and ITS 1 high-throughput sequencing technique, compare the microbiota composition between ASD children and healthy control children, and evaluate the effects of NS lactobacillus intervention on microbiota.
5.  Compare the immune indicators,  neurotransmitters  and hormones  related  to microbiota-gut-brain axis between ASD children and healthy children, explore the differences and  the influence of NS lactobacillus intervention, and  reveal the effectiveness and mechanism the intervention.
Results:
1 .ASD children not only presented obvious cognitive and behavioral symptoms,accompanied with a variety of gastrointestinal symptoms, but also they showed abnormal gut microbiota including bacteria and fungi and abnormal biochemical indicators including higher Complement C3 levels and lower GABA and Glu levels in serum.
2. NS lactobacillus intervention significantly improved gastrointestinal symptoms and ASD-like symptoms. After three months of intervention, the gastrointestinal symptoms of ASD children decreased (total GSI score significantly reduced). For example, the stool consistency improved, the stool smell alleviated, the flatulence alleviated, unexplained daytime irritability behaviors reduced, and nighttime awaking times decreased. NS intervention also significantly alleviated the severity of ASD (total ATEC scores decreased and all subscales scores reduced). For example, the language  communication,  social  interaction  and  sensory/cognitive  awareness increased, and health/physical/behavior improved after intervention.
3. NS lactobacillus intervention significantly improved the microbial imbalance of ASD children.
4. NS lactobacillus intervention significantly improved the biochemical abnormalities of ASD children. After three months of intervention, the serum Complement C3 content of ASD  children increased  significantly,  while  GABA content of that decreased significantly.
5. The improvement in physiology and behavior of NS lactobacillus intervention was correlated with the regulation of gut microbiota. For example, the Complement C3 content was negatively correlated with the relative abundance of Romboutsia and Terrisporobacter, while the GABA content was positively correlated with the relative abundance of Romboutsia and Terrisporobacter.
Conclusions:
    In summary, there were significant differences in gut bacteria and fungi between ASD and healthy control children. Abnormal gut microbiota can induce ASD through the immunological pathway and neuroendocrine pathway of microbiota-gut-brain axis.NS lactobacillus intervention reduced the gut microbiota abnormalities of ASD children, and alleviated ASD through immunological pathway and neuroendocrine pathway.
Innovations:
    The present study implemented NS lactobacillus combination intervention to the so-called incurable disease ASD for the first time. The study collected and analyzed the behavioral, physiological, biochemical, and microbiota data before and after intervention. The study firstly presented that NS lactobacillus intervention corrected the microbiota abnormalities of ASD children, improved the immune and neuroendocrine function, and alleviated the behavioral, cognitive and gastrointestinal symptoms of ASD children.

语种中文
文献类型学位论文
条目标识符http://ir.psych.ac.cn/handle/311026/26136
专题健康与遗传心理学研究室
作者单位中国科学院心理研究所
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吴晓丽. 自闭症患者肠道菌群的结构变化及NS乳酸菌干预机制研究[D]. 北京. 中国科学院研究生院,2018.
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