【作者】 郭君慧；

【导师】 杨旭； 张罗平；

【作者基本信息】 华中师范大学 ， 动物学， 2018， 博士

【摘要】 甲醛是一种重要的工业产品,能够以气体状态和溶液状态存在,通过呼吸系统或消化系统进入体内。其中,以气体状态存在的甲醛受到广泛关注。研究表明,空气中的甲醛能够导致多种病理症状,包括急性中毒症状和慢性中毒症状,危害人体健康。目前,对经呼吸系统吸入的甲醛影响人体健康的研究有很多,但是对经消化道摄入的甲醛影响人体健康的研究却非常少见。经消化系统进入体内的甲醛可能会直接作用于肠道菌群,而肠道菌群是近年来研究的热点。对于肠道菌群和健康之间的关系的研究表明,肠道菌群异常会导致许多疾病。环境污染物进入消化道后,可能会和肠道菌群相互作用,从而影响健康。然而,甲醛在进入消化道后,是否影响到肠道菌群以及如何通过与肠道菌群相互作用而导致疾病还尚未得到深入认识。因此,有必要探索甲醛与肠道菌群之间的相互作用,不仅有利于理解甲醛通过消化系统影响健康的作用机制,补充和延伸现有研究成果,也有利于理解肠道菌群与人体健康的相互关系。本论文以小鼠为研究模型,探索甲醛作用于小鼠消化系统时对于小鼠肠道菌群的影响,采用的研究方法包括传统的微生物分离和培养方法以及16S rRNA基因高通量测序与分析。对于所获得的序列数据及相关细菌菌群数据,进行了统计分析。本研究获得的主要结果如下:(1)利用微生物培养分离研究方法,对小鼠接受甲醛灌胃处理后肠道菌群的变化进行了初步研究。结果表明,小鼠接受甲醛处理后,与对照相比,并没有观察到肠道菌群的培养特性发生显著变化。将接受甲醛处理的小鼠肠道菌群样品在不同培养基、不同培养方式(有氧培养、无氧培养)下培养所得到的典型菌落进行16S rRNA基因测序以及Blast数据库分析,鉴定出17个菌属,其中绝大部分是芽孢杆菌属(Bacillus)的细菌。这些结果表明,体外培养方式仅有利于个别细菌生长。由于小鼠肠道菌群的复杂性,以及体外培养方法的缺陷,难以精确地揭示小鼠接受甲醛处理后肠道菌群的变化规律,影响了进一步的解读,以及相关的应用研究。(2)利用16S rRNA基因高通量测序分析技术,对甲醛引起小鼠明显生理中毒症状时的肠道菌群变化进行了研究。结果表明,甲醛影响肠道菌群时存在剂量效应和时间效应。在门分类水平上,拟杆菌门(Bacteroidetes)细菌和厚壁菌门(Firmicutes)细菌在所有的样本组中,相对丰度均远超过1%,是优势的细菌门类,二者合计超过肠道菌群总数的95%,远远超过其余的细菌门类,处于绝对优势地位。甲醛诱导对小鼠肠道菌群alpha多样性的OTUs(Operational Taxonomic Units)指标和PD(Phylogenetic Diversity)指标以及beta多样性产生显著影响,STAMP统计分析、热图结合聚类分析等结果表明,肠道菌群在甲醛处理中部分微生物种类的相对丰度发生显著变化。基于PICRUSt程序对甲醛处理的小鼠肠道菌群功能进行了预测,结果表明,部分代谢途径如 alpha 亚麻酸代谢(alpha-Linolenic acid metabolism)、醚酯代谢(Ether lipid metabolism)、辅酶和维生素代谢(Metabolism of cofactors and vitamins)、幽门螺旋杆菌感染相关的上皮细胞信号途径(Epithelial cell signaling in Helicobacter pylori infection)、转录机(Transcription machinery)的丰度发生显著变化。上述结果表明,在引起小鼠中毒症状时的甲醛处理下,小鼠肠道菌群的种群结构与功能均发生明显变化,这些变化有可能参与甲醛导致的中毒生理反应。(3)当较低剂量甲醛短期作用于小鼠,小鼠还没有发生明显中毒症状或者仅发生轻微中毒症状时,同样进行16S rRNA基因高通量测序与分析。结果表明,小鼠肠道菌群结构在接受甲醛或甲醇处理后7天和14天趋于稳定。利用STAMP统计分析和热图结合聚类分析,在门分类水平和属分类水平对小鼠肠道菌群中对于甲醛和甲醇敏感的细菌进行了检测,其中对于甲醛处理敏感的菌包括Prevotelh属、Streptococcus属、Lactococcus属、Candidatus Arthromitus属、Delftia属。推测存在一定的修复或应激机制,保护小鼠肠道菌群抵御早期低剂量甲醛的影响。(4)本研究还发现生理盐水的作用能够导致小鼠肠道菌群相对丰度发生变化,并在门分类水平和属分类水平检测出几种受到生理盐水影响的细菌,包括脱铁杆菌门(Deferribacteres)细菌、厚壁菌门(Firmicutes)细菌和拟杆菌门(Bacteroidetes)细菌。由于这种变化是相对比较稳定的,可以推测,生理盐水处理后,小鼠肠道菌群能够处于一个比较稳定的状态。综上所述,本研究探索了甲醛处理对小鼠肠道菌群的影响,提供了小鼠肠道菌群结构和丰度在不同实验条件下的数据与分析结果。结果表明,高剂量甲醛暴露对于肠道菌群的物种结构和功能可以产生显著影响,其中部分显著性差异微生物物种可能参与甲醛影响健康的过程。这项扩充了有关甲醛毒理学研究领域,并为有关甲醛、肠道菌、以及人体健康的进一步研究提供了有益指导。更多还原

【Abstract】 Formaldehyde is an important industrial product that exists in either gas or solution states.It enters the body through the respiratory or digestive system.Many studies have shown that air formaldehyde can cause pathological symptoms,including acute poisoning symptoms and chronic toxic symptoms,especially cancers that harm to human health.Although there are many studies on effects of air formaldehyde inhalation on human health through the respiratory tract,the study on the intake of formaldehyde through the digestive tract is rare.Intestinal microflora has attracted lots of scientists in recent years.There have been a number of studies on the relationship between the intestinal microflora and health,especially the changes with the intestinal microflora,which can lead to many diseases.Environmental pollutants entering the digestive tract and will firstly interact with the intestinal microflora,and then affect health.Up to date,only a few studies have shown that environmental pollutants can affect the intestinal microflora.Therefore,exploring the interaction"between formaldehyde and intestinal microbiota is not only helpful in understanding the mechanism of formaldehyde in affecting the digestive system,but also complementing and extending the existing studies.It is also helpful in understanding the relationships between intestinal microflora and human health.In this dissertation,mice were used as a research model to study the dose and time effects of formaldehyde on the mouse digestive system.The research methods included traditional microbial isolation and culture methods,and 16S rRNA genes high-throughput sequencing and metagenomic methods.For the sequence data obtained,as well as the microbial microflora data generated,further statistical analysis and statistical comparison using software such as STAMP.The important results obtained are as follows:Firstly,a preliminary study was performed on the changes of intestinal micro:flora in mice before and after the treatment of formaldehyde treatment by microbiological culture.The results showed that after the mice receiving formaldehyde treatment,no significant changes in the culture characteristics of the intestinal flora were observed.The typical colonies of intestinal microflora in mice receiving formaldehyde treatment were analyzed by 16S rRNA genes sequencing after cultivation by using different medium and different culture methods(aerobic culture and anaerobic culture).The effective sample data,by Blast analysis,17 bacteria were found,most of which were bacteria of Bacillus Bacillus.These results indicate that in vitro culture methods are conducive to the growth of individual bacteria.Therefore,due to the complexity of intestinal microflora in mice and the defects of in vitro culture methods,it is difficult to accurately reveal the changes of intestinal microflora in mice after formaldehyde treatment,affecting further interpretation and related application researchSecondly,by using 16S rRNA genes high-throughput sequencing combined with metagenomic analysis technology,formaldehyde-mediated changes in intestinal microflora in mice were studied.It was initially revealed that when formaldehyde can cause symptoms of poisoning in mice,formaldehyde treatment for intestinal bacteria has the dose effects and time effects.Using high-throughput sequencing,the abundance and distribution of mouse intestinal bacteria were obtained at the level of phylum,families,and genera.The results showed that the relative abundance of Bacteroidetes and Firmicutes bacteria in all groups far exceeded 1%,which was the dominant bacteria class.Both of them exceeded the 95%of total number of gut bacteria,is far more than the rest of the bacteria and is in an absolute dominant position.Using STAMP statistical analysis and heat map combined with cluster analysis,the changes of intestinal bacterial abundance caused by formaldehyde treatment were analyzed.The PICRUSt program was then used to predict the effect of formaldehyde treatment on the function of the intestinal microflora in mice in order to reveal the dose and duration of formaldehyde treatment that can cause significant poisoning in mice.The results showed that formaldehyde induction could not fundamentally affect the structure and composition of intestinal microflora in mice,but it can induce significant changes in the relative abundance and diversity of intestinal microflora,especially in the taxonomic and family classification levels.The relative abundance and diversity of bacteria have changed significantly.The bacteria that are affected include not only the dominant families in the intestinal microflora,but also the genus bacteria,as well as rare families and genus bacteria.By predicting KEGG metabolic pathways and correlation analysis,it was found that formaldehyde induction can cause a series of related detoxification metabolic pathways and signal pathway changes in the intestinal microflora.Thirdly,after short-term(7 or 14 days)and lower doses of formaldehyde exposure,the mice have not developed symptoms of significant poisoning or only mild symptoms of poisoning,the 16S rRNA high-throughput sequencing results showed that intestinal microflora in mice was relatively stable after 7 days of formaldehyde treatment or 14 days of formaldehyde treatment.Using STAMP statistical analysis and heat map combined with cluster analysis,several bacterial being senstive to formaldehyde treatment were detected including Prevotella,Streptococcus,Lactococcus,Candidatus Arthromitus,Delftia.It is speculated that there is a certain repair or stress mechanism,which can protect the intestinal microflora of mice and resist the effect of early low dose formaldehyde,however,with the prolongation of formaldehyde action time,this protective mechanism is suppressed or lost,resulting in intestinal microflora damage.Finally,the present study also found that the effect of physiological saline can lead to changes in the relative abundance of intestinal microflora in mice,and detected several bacteria that were affected by physiological saline at the level of gate and genus classification.Since this change is relatively stable,it can be speculated that after saline treatment,the intestinal microflora of mice can be in a relatively stable state.This finding provides guidance for future research on the changes of intestinal microflora induced by environmental pollutants.In summary,this study explored the effects of formaldehyde exposure on intestinal microflora in mice and firstly provided the data on the effects of formaldehyde impact on the structure and abundance of intestinal microflora in mice.The results of the study showed that although there was no fundamental change in the structure and composition of the intestinal microflora,formaldehyde exposure had an impact on the relative abundance of intestinal micro:flora,and identified a number of dominant bacteria,common bacteria,rare bacteria and bacteria that were affected by formaldehyde treatment,and have expanded their research fields in the field of formaldehyde toxicology,providing guidance for further research on formaldehyde,intestinal bacteria,and human health.更多还原