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题名:
菊粉对小鼠肠道微生物调节作用的研究
作者: 朱立猛1,2
学位类别: 硕士
答辩日期: 2017-05-15
授予单位: 中国科学院大学
授予地点: 北京
导师: 秦松
关键词: 菊粉 ; 肠道微生物 ; 肥胖 ; 炎症 ; 宏基因组
学科分类: 地球科学::海洋科学::海洋生物学
中文摘要:     肠道是一个极其复杂的生态系统,是1000万亿厌氧微生物的寄居地。肠道菌群对于维持人体健康具有重要的意义。肠道菌群失调是肥胖、糖尿病等多种慢性疾病的重要诱因。研究表明,除了自身基因型的影响外,环境因子是影响肠道菌群组成和结构的重要因素,其中饮食结构尤为重要。近年来,肥胖的发生率在世界各地迅速上升,成为威胁到人类健康的普遍问题。肥胖的发生往往伴随着慢性、低度的炎症,属于肥胖导致的代谢并发症。随着对肠道菌群相关研究的日益深入,越来越多的研究结果表明肠道菌群与肥胖及其并发症的发生有着密切的关系,肠道菌群作为环境因子影响肥胖发生的科学问题越来越受到研究者们的关注,但相关的的机制依旧不明确,确切的机理仍在研究中。
    菊粉作为一种益生元,由于其特殊的β-(2,1)-糖苷键结构,使得菊粉在口腔、胃、小肠中均不会发生消化分解,只能在结肠中被肠道微生物作为底物分解产生少量的热量,这一特殊的性质使得菊粉具有了许多突出的生理功能。菊粉的摄入能够选择性的促进益生菌的增长,抑制有害菌的增殖。然而菊粉对肠道菌群的影响非常复杂,先前的研究往往只关注了特定的种属,而对肠道菌群缺乏全面的理解。高通量测序技术的飞速发展,使我们对肠道菌群组成和结构的深入了解成为可能。
    我们首先利用16s rRNA扩增子测序的技术手段,研究了不同聚合度的菊粉对健康小鼠肠道菌群组成和结构的影响。结果显示菊粉干预后LactobacillaceaeBifidobacteriaceae的相对丰度都显著增加。但菊粉对于肠道菌群的影响不仅仅局限于双歧效应,Bacteroidales S24-7 groupRuminococcaceae等丁酸盐产生菌的相对丰度也显著增加。Akkermansia muciniphilaVerrucomicrobia门中定殖在黏液层中的黏蛋白(mucin)降解菌,其相对丰度在菊粉干预后也显著增加。菊粉对肠道菌群的调节作用与聚合度(degree of polymerization, DP)和剂量密切相关。短链菊粉在促进益生菌增殖方面展现出了更好的效果;在高剂量处理组中,肠道益生菌相对丰度的增长幅度往往更大。Bacteroides vulgatus与其它菌群之间的互作现象也在该研究中被证实。基于对结果的分析,我们认为Akkermansia muciniphila可能是维持mucin和短链脂肪酸(short chain fatty acids, SCFAs)动态平衡的关键因子。
    为了研究短链菊粉对高脂饮食诱导产生的肥胖型小鼠肠道菌群的影响。我们基于Illumina Hiseq PE250的测序平台,采用16S rRNA V4和V5高变区高通量测序的技术手段,全面深入分析5 %的短链菊粉添加对肥胖小鼠肠道菌群的影响。研究结果显示,高脂饮食诱导产生的肥胖小鼠体内Firmicutes/Bacteroides的比例显著上升,Bacteroidales S24-7 groupRuminococcaceae等丁酸盐产生菌的相对丰度显著下降,Escherichia-ShigellaParabacteroidesBacteroides等革兰氏阴性条件致病菌的相对丰度显著增加。而短链菊粉的摄入,能够显著降低Firmicutes/Bacteroides的比例,促进肠道内益生菌相对丰度显著增加,同时能够减少条件致病菌的数量。
    为了进一步研究短链菊粉通过调节肠道菌群影响肥胖及其炎症并发症的发生过程。我们利用实时荧光定量PCR的分析手段,对小鼠肝脏和附睾脂肪垫中的炎性因子相关基因及脂代谢相关基因的表达情况进行了分析。结果显示在高脂饮食组中,炎性因子相关基因及脂代谢相关基因都呈现出高表达的状态,瘦素(leptin)水平显著升高,机体出现了leptin抵抗的现象。而短链菊粉的添加,使得炎性因子和脂代谢相关基因的mRNA表达水平较高脂饮食组都发生了显著下降。
脂多糖(lipopolysaccharides, LPS)是引发肥胖患者全身性慢性炎症的诱因。基于研究结果,我们提出短链菊粉能够调节肥胖及炎症反应的可能性机制:一方面因其益生菌保护效应能有效调节肠道菌群的结构,促进有益菌相对丰度增加,相应的抑制产LPS的革兰氏阴性条件致病菌的增殖,增强肠黏膜屏障功能。另一方面,短链菊粉在肠道内的发酵产物SCFAs不但可以降低肠道微环境的pH值,抑制有害菌的增殖,减缓炎症的发生;还能够有效调节机体能量代谢,减缓肥胖的发生过程。该研究对于全面深入解析菊粉对肠道菌群的调节作用,了解肥胖发生及其预防治疗有一定的指导意义。
英文摘要:   The human intestine is inhabited by a highly diverse microbial ecosystem dominated by hundreds of different species of anaerobic bacteria. The complex bacterial community plays a critical part in biological processes. Intestinal microbial community dysbiosis is the leading cause of numerous chronic diseases. It has been clearly demonstrated that environmental factors, particularly diet, have a considerable effect on the composition of intestinal microbiota besides the individual genotype. Obesity, accompanied by inflammation, has become a worldwide devastating epidemic in recent decades. Recent studies on intestinal microbiota have revealed the causative role of the gut microbial dysbiosis in obesity. However, the exact mechanisms are still under studies.
  Inulins are a polysaccharide linked by β-(2-1) bonds through D-fructoses with a terminal glucose residue. And inulins can pass through the upper gut and reach the large intestine unabsorbed, modulates the intestinal microbial community, leading a beneficial effect on host. Inulins have been reported to stimulating growth or activity of beneficial bacteria and suppressing the levels of harmful bacteria selectively. However, influences of dietary inulin on intestinal microbiota were very complex. The effects of inulin on modulating intestinal microbiota were mainly concerned on given groups of bacteria during inulin consumption in previous studies. And little research to date has been focus on global gut microbiota communities during interventions of inulin. The high-throughput sequencing technologies have profoundly changed our understanding on diversity of intestinal microbiota.
  The study aimed to analyse the global influences of dietary inulin with different degrees of polymerization (DP) on intestinal microbial communities by 16s rRNA sequencing technology. A direct consequence of inulin interventions is stimulation of Bifidobacteriaceae and Lactobacillaceae within intestinal microbiota, which is called bifidogenic effect. However influences of dietary inulin on modulating intestinal microbiota were far more complex than bifidogenic effects. The relative abundance of butyrate-producing bacteria, within Bacteroidales S24-7 group and Ruminococcaceae families were increased after FOS and inulin interventions. Akkermansia muciniphila, belonging to mucin-degrading species, became a dominant species in Verrucomicrobia phylum after inulin treatment. The modulation effects of  inulin were positively correlated with DP and dosage. Lower DP interventions exhibited better effects than higher DP treatment on stimulation of probiotics. The increasing extent of some gut microbiota was always stronger in high dose groups than in other groups. The increasing abundance in Bacteroides vulgatus, which cannot use inulin, indicated a cross-feeding phenomenon. And we hypothesized that Akkermansia muciniphila played an important role on maintaining balance between mucin and short chain fatty acids (SCFAs).
  In order to investigate the influences of dietary FOS on intestinal microbial communities in high fat diet (HFD) induced obesity mice, the V4 and V5 hypervariable 16S rRNA region were selected for sequencing based on Illumina Hiseq PE250 paltform. The ratio of Firmicutes to Bacteroides increased significantly in HFD group. The relative abundance of butyrate-producing bacteria, within Bacteroidales S24-7 group and Ruminococcaceae families were decreased in HFD group. And gram-negative opportunistic, endotoxin producing pathogens, such as Escherichia-ShigellaParabacteroides and Bacteroides, increased significantly in HFD group. FOS intervention decreased the ratio of Firmicutes to Bacteroides. An increase in probiotics and a decrease in opportunistic pathogens were also found with FOS supplement.
  Real-time quantitative PCR was used to identify the expression level of lipometabolism and inflammation related genes in liver and adipose. The excessive fat accumulation in the HFD-fed mice was associated with a leptin-resistant phenotype. And the expression level of mRNA increased significantly in HFD group. However, FOS intervention decreased the level of mRNA compared with HFD group.
  The level of lipopolysaccharides (LPS) may be the leading cause of obesity and inflammation. We hypothesized that the mechanism of FOS on reducing obesity and inflammation: on the one hand, FOS could modulate the structure and composition of intestinal microbiota, as well as enchance the function of intestinal barrier. Furthermore, FOS could stimulate the growth or activity of beneficial bacteria and suppress the levels of gram-negative opportunistic, endotoxin producing pathogens selectively. On the other hand, the SCFAs, which were generated by intestinal microbiota fermentation, could have reduced pH in micro-environment of intestinal tract, as a consequence of suppressing the levels of opportunistic pathogens. Besides, SCFAs could modulate energy metabolism, reducing the process of obesity. This study has a guiding effect for inulin on modulating intestinal microbiota in mice, as well as understanding the occurrence and prevention of obesity.

 
内容类型: 学位论文
URI标识: http://ir.yic.ac.cn/handle/133337/22451
Appears in Collections:中科院烟台海岸带研究所知识产出_学位论文

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作者单位: 1.中国科学院烟台海岸带研究所
2.中国科学院大学

Recommended Citation:
朱立猛. 菊粉对小鼠肠道微生物调节作用的研究[D]. 北京. 中国科学院大学. 2017.
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