【作者】 吴韬；

【导师】 张捷；

【作者基本信息】 昆明医科大学 ， 外科学， 2014， 博士

【摘要】 背景：细菌与胆固醇结石发生的相关性一直存在争议；关于胆道菌群的组分、结构和起源,受限于研究的条件以及测序的高成本,检测出来的细菌的种类和数目极为有限,缺乏全面完整的概念；胆囊结石患者是否存在肠道菌群失调仍然未知；目前关于细菌与结石形成的相关性的研究,多局限在体外模拟实验及培养的条件下对生理生化上的研究,缺乏细菌功能基因的研究。目的：本研究拟从肠道菌群和胆道菌群出发,借助新兴的二代测序技术,全面系统的揭示并比较胆结石患者的胆道(胆结石和胆汁)菌群、肠道菌群的结构和组分的变化规律；探讨胆结石患者是否存在肠道菌群失调,鉴定与结石病发生相关的胆道核心菌群,拟从细菌的功能角色层面(或功能基因层面)探讨与胆结石发生的相关性,为进一步研究胆固醇结石形成的细菌学机制提供重要的素材和前期工作基础,为临床上防治胆结石提供一定的参考。方法：本研究中胆道菌群采用胆囊结石患者的胆结石及胆汁的菌群为代表,胆结石患者的肠道菌群采用其粪便菌群代表,肠道的正常菌群采用正常人的粪便菌群作为代表。我们收集了29例胆囊结石患者的结石、胆汁和粪便,并收集了38例正常人的粪便共120个样本。主要方法有：1.采用酶CHOD-PAP法,测定胆囊结石的胆固醇含量并鉴定胆囊结石的类型；2.于非培养条件下,采用物理破碎和化学裂解相结合的方法,提取样品的细菌DNA,采用酶链聚合反应(PCR)进行扩增。PCR扩增产物经过电泳分析、胶回收、纯化；3.采用高通量的454焦磷酸测序技术对16S rRNA基因V1-V2可变区的PCR扩增子进行大规模的测序,获取序列数据,鉴定细菌OTU(相当于种的水平)；4.结合生物信息学、网络分析和功能注释相似的物种的基因组的手段,与已公布的细菌基因库比对,并结合统计学的方法检查和分析从所有胆结石患者所获得的胆汁、胆石及粪便,以及正常人粪便的细菌组分、群落结构。结果：1.本研究中胆固醇结石所占比例为92.55%,代表了胆囊结石的主要类型。2.所有类型的样本(包含纯胆固醇结石)均检测出细菌DNA,检出率为100%。3.本研究从所有样总计获得299,217条高质量、可分类的细菌16SrRNA基因序列。其中,从胆结石患者的胆汁中获得81661条细菌基因系列、结石中获得76135条细菌基因系列、粪便中获得64360条细菌基因系列；从正常个体的粪便共获得77601条细菌基因系列。4.共鉴定了4637个细菌OUT。其中,在患者的胆汁中鉴定了3696个细菌OTU、结石中鉴定了3456个细菌OTU、粪便中鉴定了1772个细菌OUT；正常个体粪便中鉴定了1497个细菌OUT；所鉴定的细菌OTU能够被划分为20种细菌门类群,胆结石中有20种,胆汁中有19种,在胆结石患者的粪便中有13种,在正常个体的粪便中有14种。最占优势的细菌类群为厚壁菌门。5.胆道菌群比较(胆汁和结石)：胆汁与结石菌群无明显差异(P>0.05),6.肠道菌群比较(正常人和胆囊结石患者)：(1)细菌门的水平：胆结石患者的肠道细菌中Proteobacteria(变形菌门)较正常人显著增高(P<0.001)；(2)细菌属的水平：三个肠道细菌属Faecalibacterium(柔嫩梭菌属),Lachnospira(毛螺菌属),Roseburia(氏菌属)较正常人显著减少(P<0.001)。7.胆囊结石患者肠道菌群和胆道菌群比较：(1)细菌门的水平：最占优势的细菌门Firmicutes以及最为稀少的细菌门Fusobacteria,二者均无明显差异(P>0.05)；除了Bacteroidetes在胆道中的组成显著低于肠道外(P<0.001),其余的六个细菌门类Proteobacteria、Tenericutes、Actinobacteria、Cyanobacteria、TM7、Thermi在胆道中的组成均显著高于肠道(P<0.001)；(2)细菌属的水平：胆道的两个细菌属Bacteroides和Clostridium(L.)较肠道显著减少(P<0.001),而另外16个细菌属较肠道显著增加(P<0.001)；胆道菌群的多样性显著高于肠道菌群(P<0.001)。8.胆汁和结石分享超过85%的细菌OTU；仅60%的肠道细菌OTU在病人和正常人之间分享；约有70%的病人肠道细菌OTU以及正常人40%的肠道细菌OTU在胆道中被发现。9.鉴定并分析了胆囊结石患者胆道核心菌群(包括106个细菌OTUs),仅33.96%(36/106)能匹配到已知的细菌种类,而其中只有15种细菌具有已知的已公布的基因组。这15种胆道细菌至少包含了四个基因(MDR外排泵蛋白,BSH, bG和phL,被认为是促结石形成的四种细菌因子)中的一个。结论：1.本研究首次采用了高通量的二代测序的方法(454焦磷酸测序),通过大规模的分析胆道和肠道细菌的16SrRNA基因,首次从系统层面全面的揭示了胆道细菌群落多样性和结构组分的基本特征。2.本研究所发现的结石和胆汁的细菌的种类和数量均远远超出以往的发现,极大的丰富和拓宽了胆道的细菌学谱,为后续研究提供了一定的基础。3.本研究首次揭示了胆囊结石患者存在明显的肠道菌群失调,提示了胆结石患者存在潜在的胃肠道健康风险,另一方面也提示了肠道菌群失调也可能导致胆囊结石形成；胆道菌群至少部分源于肠道菌群的移位,且比肠道菌群更加复杂多变。4.本研究揭示了胆道的核心菌群,并鉴定了核心菌群中与结石形成相关的四种细菌因子(MDR外排泵蛋白、bG和phL),胆道核心菌群与胆结石的形成密切相关。5.胆结石形成的细菌学机制可能为细菌-细菌的综合多因素共同作用。6.本研究对胆结石形成相关的细菌学机制作了较为全面的探讨,充分探讨了细菌及菌群失调与胆结石形成的关系,对理解胆囊结石形成的细菌学机制做了有益的补充,对临床上对胆结石,尤其是胆固醇结石的预防和治疗提供了一定的参考。更多还原

【Abstract】 Objective:The aims of this study are:1) to reveal the variation of bacterial community and composition in both gut and biliary tract (gallstones and bile) in patients with gallstones;2) clarify whether there exist gut bacterial dysbiosis in patients with gallstones;3) to identify the biliary tract bacteria associated with the formation of cholesterol gallstones, as well as to elucidate the relationship between the biliary tract bacteria and cholesterol gallstones formation out of four potential bacterial factors contributing to cholesterol gallstones. The study will provide foundationalfindings for further studying bacteriological mechanism of the cholesterol stone formation, which will be helpful for both preventions and therapeutics for cholelithiasis.Methods:We collected samples of stone, bile and feces from29patients with gallbladder stones, and also fecal samplesfrom38normalindividuals, and finally obtained a total of120samples. The content of cholesterol in gallstones was analyzed for the classification of gallstone disease. The bacterial16S rRNA gene was amplified by PCR methods using bacterial DNAs extracted from all120samples and was used to perform454-barcoded pyrosequencing. Bacterial16S rDNA sequences were filtered and used to identify bacterial OTUs (equal to bacterial species) according to97%sequence identity. Final OTUs were used to clarify thecommunity structure and composition of bacteria in both gut and biliary tractwith cholelithiasis. Based on these results, a series of analyses were further performed, including heat-map based abundance, network analysis, and functional annotation depending on similar species’ genomes.Results:1. Cholesterol stone proportion is92.55%of all sampled stones, which represents the main type of gallstone disease.2. Bacterial DNAs were detected from all samples.3.We obtained a dataset consisting of299,217high-quality and classifiable16S rRNA gene sequences. From the dataset, we identified a total of4637operational taxonomic units (OTUs). These identified bacterial OTUs can be classified into20bacterial phyla.4. Comparison of gutbacteria community (normal individuals and patients with gallbladder stone) was conducted. Within the gut, the relative abundance of the bacterial phylum Proteobacteria among patientswere significantly (P<0.001) higher than that in normal. The relative abundances of three bacterial genera (Faecalibacterium, Lachnospira,andRoseburia) in normal subjects were significantly (P<0.001) lower in those in patients.5. Gut bacteria were compared with biliary bacteria in the gallstone patients.(1) This study found that Bacteroidetes phylum in biliary tract was significantly lower than that in the gut (P<0.001).The abundances of six bacterial phyla in the biliary tract, includingProteobacteria, TM7, Tenericutes, Actinobacteria, Thermi,and Cyanobacteriawere significantly higher than those in the gut (P<0.001).(2) This study also found that, in the biliarytract, there was a significant (P<0.001) decreasing of two bacterial genera and increasing of16otherbacterial genera (P<0.001). Bacteriadiversity in the biliary tract was significantly higher than that in the gut (P<0.001).6. This study showed that over85%of bacterial OTUs were shared by both bile and gallstones, but only60%ofgut bacterial OTUs were shared by patients and healthysubjects. Around70%of gut bacterial OTUs from patients and40%OTUs from healthy subjects were found in the biliary tract, respectively.7. Based on the dataset described above, this study identified biliary tract core microbiome out of gallstone patients, which includes106bacterial OTUs belonging to six bacterial phyla. Only33.96%(36/106) of the members of the biliary tract core microbiomecould be matched to known bacterial species. About15of the36-matched bacterial species havepublicly available genomes. Based on reference genomesof those15bacterial species, this studyfurther analyzed the presence or absence of four genes potentially associated withthe formation of gallstones, includingMDR, BSH, bG,andphL.The results found each of15speciesat least contained one of the four genes.Conclusions:1. This is the first study to use a454-basedpyrosequencing of bacterial16S rDNAs to clarify the structure and composition of bacteria in both gut and biliary tract with cholelithiasis.2. To the best of our knowledge, this is the first study todiscover a potential association of the gut microbiotadysbiosis that is present among gallstone patients.Our results discovered significant changes of gut microbial components between gallstone patients andhealthy subjects. This raises a possibility that biliary tractmicrobiota could be partially originated from the gut. Furtherevidence is obviously needed to explore this possibility,but nonetheless it is an intriguing prospect. 3. This study supports mounting evidence that cultureindependent454-based16S rDNA sequencing is necessaryas a diagnostic tool in gallstone-associated bacterial infection, because it provides more detailed information thancurrently employed and sequence analysis of clonedmicrobial16S rDNA.4. This study reveals the core of biliary tract bacteria and identified four types of bacteria associated with stone formation factors (MDR efflux pump protein, BSH, bG and PhL). The biliary core bacteria are closely related to the formation of gallstones.5. These findings indicatedthat bacterial community assembly might be more important than single species in the formation of cholesterol gallstones.6. This study makes a comprehensive discussion of the relationship between the bacteria community assembly and cholesterol stone formation. These findings have numerous medical implications for both prevention and therapeutics for cholelithiasis or other relative GIT healthy risks, warranting further follow-up studies that are needed to verify these findings and move forward. The most poignant implication of this study is that prevention and management of bacterial infections in the biliary tract may be a target for lowering the risks of cholesterol gallstones. The study also provides some clues for further exploringthe mechanism of bacteriology leading to the cholesterol stone formation.更多还原