影响小鼠肠道微生态的环境因素研究

【作者】 杨帆；

【导师】 周东蕊；

【作者基本信息】 东南大学 ， 生物医学工程（学习科学）， 2020， 硕士

【摘要】 肠道微生物结构组成与多样性和免疫系统疾病发生密切相关,探索肠道微生物影响因素具有重要研究意义。卫生假说指出低清洁度环境对免疫系统疾病有显著保护作用。本课题组前期研究发现,清洁度较低环境中生活的小鼠肠道微生态丰富度与多样性较高,并发现环境中仅仅增加微生物,对小鼠肠道微生物的多样性影响较小,而环境中增加土壤对小鼠肠道微生物结构组成与多样性有显著影响。土壤中含有丰富的微生物,其种类约为肠道微生物类型的10倍,被誉为“微生物种质银行”,环境中的土壤是以什么途径影响肠道微生物的结构组成,是该项研究拟解决的关键科学问题。我们提出环境中的土壤通过食入体内方式为主来影响肠道微生物这一假设,并以小鼠为研究模型进行探究。将来源一致的38只小鼠分为四组,分别为:高清洁环境对照组(SPF)组,饲喂有菌土壤组(Farm),饲喂高压灭菌土壤组(SS),饮用环境微生物组(MW)。同时,我们还采用2-4-二硝基氟苯(DNFB)处理来验证土壤通过改变小鼠肠道微生物结构对小鼠免疫力的影响。实验内容:将38只7周龄SPF级别C57小鼠随机分为四组,小鼠年龄、窝别、性别、分娩方式和前期饲养条件等都控制一致。其中SPF、SS、MW组饲养在同一间SPF动物房,Farm组饲养在另一间SPF动物房中。SPF组为对照组,SS组饲料中添加灭菌土壤,MW组饮用从土壤中分离出的微生物,Farm组饲喂一定比例未灭菌土壤,其它条件都控为一致。处理42天后收集小鼠粪便,并进行16S r RNA基因测序和宏基因组测序进行肠道微生物分析。第43天,我们开始采用DNFB对小鼠皮肤过敏处理,通过血清总Ig E检测和皮损程度分析,来评测每组小鼠肠道微生物过敏保护功能。实验结果:(1)食用有菌土壤组(Farm)小鼠与食用无菌土壤组(SS)小鼠肠道微生物的多样性和丰富度显著高于饮用环境微生物(MW)和对照组小鼠(SPF),Farm组多样性和SS组没有显著差别,MW组多样性和SPF组也没有显著差别;(2)SS小鼠与Farm小鼠肠道微生物β多样性更为相似,而MW小鼠和SPF小鼠β多样性相似度更高。(3)宏基因组测序结果显示,SS小鼠含有更丰富的III型分泌系统编码基因,Farm小鼠除含有丰富的III型分泌系统编码基因外,还富含短链脂肪酸和氨基酸合成酶。(4)DNFB处理结果显示,SS与Farm小鼠免疫力显著高于另外两组,MW组和SPF组差异不显著。结论:食入土壤是影响小鼠肠道微生物结构组成与多样性的一种重要方式。该研究也进一步验证,饮用大量环境中的微生物对肠道微生态结构组成有明显的影响,但对肠道微生物多样性影响较小。更多还原

【Abstract】 Gut microbial diversity and structural composition are closely related to the occurrence of allergic diseases and it is great significance to explore the influencing factors of intestinal microbes.The hygiene hypothesis indicates that low environmental cleanliness(e.g.farms)has a significant protective effect on allergic diseases.Our previous study found that mice living in low cleanliness environment had higher intestinal micro-ecological richness and diversity,and found that only increasing microbes in the environment had little effect on intestinal microbes in mice,while increasing soil in the environment had a significant effect on intestinal microbial structure composition in mice.Soil is rich in microorganisms,the species of which is about 10 times the type of intestinal microbes,known as the "microbial germplasm bank ".What ways does the soil in the environment affect the structural composition of intestinal microbes is the problem to be solved in this study.The soil in the environment can further affect the gut microbiota by affecting the mouse skin microbiota.It has been found that the microbial structure of dogs living in different cleanliness environments is quite different.Soil in the environment can also affect the gut microbiota by feeding into the body.Soil maybe also affect gut microbes simultaneously through two pathways.Intestinal microecology is closely related to immune system diseases and it is great significance to explore the influencing factors of intestinal microecology.Previous studies of our group found that exposure to lower environment cleanliness increases gut microbial diversity of mouse.To explore this issue 38 mice with consistent sources were divided into four groups in this study: high-definition environmental control group(SPF),low-clean environmental control group(Farm),fed high-pressure sterilized soil group(SS),and drinking environmental microbial group(MW).At the same time,we also used the DNFB eczema model to verify the correlation between intestinal microbiota and immunity in mice.Experiment content: 38 C57 mice of the same 7 weeks old SPF grade were randomly divided into four groups(SPF,Farm,MW and SS).The mouse had the same age,litter,sex delivery mode and pre-feeding conditions.SPF,SS and MW group continued to be kept in SPF animal houses1;Farm group were kept in the SPF animal house2.SPF group added sterile bedding and fed normal feed and drinking water.SS group added sterile bedding and fed sterilized soil and common feed mixture.MW group added sterile bedding and drank the water of microorganisms isolated from the environment.Farm group bedding mixed 1/3soil and the mouse fed normal water and food.Four groups of mice were fed water and food once a week.After 42 days of treatment,mouse feces were collected and 16 S r RNA sequencing and macrogenome sequencing were performed for gut microbial structure composition analysis.On day 43,we began to use 2-4-dinitrofluorobenzene(2,4-dinitrofluorobenzene,DNFB)for allergic skin irritation in mice,and measured the protective function of intestinal microorganism in each group by serum total Ig E detection and skin lesion degree analysis.Results:(1)The diversity and richness of intestinal microbes in farm house group(Farm)and sterile soil group(SS)mice were significantly higher than those in drinking microbe water group(MW)and Clean environment group(SPF).(2)The gut microbial βdiversity of SS group were more similar to Farm group,while MW and SPF were more similar.(3)The results of macrogenome sequencing showed that SS group contained more type VI secretion system coding genes,Farm mice contained more type III secretion system coding genes.(4)DNFB treatment results showed that immunity of SS and Farm mice improved significantly.Conclusion: Feeding into the soil is a way to influence the gut microbial structure of mice.This study also further verified that the simple increase of microbial content in the environment had little effect on the diversity of intestinal microecology.更多还原