【作者】 徐欣；

【导师】 祝荫； 谢川；

【作者基本信息】 南昌大学 ， 临床医学硕士（专业学位）， 2023， 硕士

【摘要】 背景及目的:急性胰腺炎(acute pancreatitis,AP)是消化系统的常见疾病,其中约20%发展为重症急性胰腺炎(severe acute pancreatitis,SAP),SAP患者病程早期易出现器官功能障碍,病死率可达15-20%,若病程后期出现感染性并发症,则病死率高达30%。肠源性感染被认为是AP患者感染性并发症发生的主导因素,已有相关研究在AP患者的胰腺坏死引流液和外周血中检测到肠道条件致病菌,表明它们可能通过受损的肠黏膜屏障进入循环系统和腹腔组织器官,造成肠源性感染。然而,目前尚无研究通过同时留取粪便、胰腺坏死引流液、血液及深部痰液的样本进行分析,以直接确定AP患者不同部位菌群与肠道菌群的相关性。基于此,本研究旨在分析AP患者首次行胰腺坏死微创干预时的胰腺坏死穿刺液、血液及深部痰液的菌群,分析其与肠道菌群的同源性,进一步验证AP的肠源性感染理论。方法:本研究前瞻性地纳入2021年10月至2022年3月收入南昌大学第一附属医院消化科住院的AP患者,在患者首次行胰腺坏死微创干预时,同时留取胰腺坏死穿刺引流液、血液、粪便和深部痰液的标本,基于Pac Bio SMRT测序技术,进行全长16S r DNA测序,分析入组患者的胰腺、血液、肠道及肺部的菌群结构特征,比较各部位菌群与肠道菌群的同源性。收集入组患者的临床资料,分为感染性胰腺坏死(Infected pancreatic necrosis,IPN)组和无菌性胰腺坏死(Sterile pancreatic necrosis,SPN)组,比较两组之间肠道菌群的差异,以及两组胰腺、血液、肺部的菌群与肠道菌群的相关性。结果:1.本课题共纳入研究对象22例,包括IPN组患者16例和SPN组患者6例,两组之间性别、年龄、AP病因、首次微创干预方式、发病至入院时间和发病至首次微创干预时间天数等基线资料均无统计学差异。2、IPN组与SPN组肠道菌群的组成特点与比较:α多样性显示,IPN组肠道菌群的物种多样性指数如Chao1指数、Shannon指数有低于SPN组的趋势,但差异不具有统计学意义(P>0.05)。基于bray-curtis距离的PCo A分析显示,IPN组和SPN组的肠道菌群结构存在显著差异(Adonis检验,P<0.05)。进一步行物种组成分析,结果显示在门水平,IPN组有益菌门疣微菌门的相对丰度较SPN组显著减少(0.9%比12.6%;P<0.05);在种水平,IPN组肠道菌群中条件致病菌大肠杆菌以及罗尔斯顿菌、莱迪氏鞘氨醇单胞菌和柄杆菌的相对丰度较SPN组显著增多(P<0.05)。进一步通过q PCR技术验证两组粪便样本中大肠杆菌的表达丰度,结果表明IPN组粪便大肠杆菌的相对丰度显著高于SPN组(P<0.05)。3、IPN组与SPN组的肠道-胰腺菌群相关性:对IPN组和SPN组的胰腺菌群进行物种组成分析,在门水平,两组胰腺菌群均主要以变形菌门为主。在种水平,IPN组胰腺的优势菌群为粘质沙雷菌、罗尔斯顿菌、大肠杆菌、戴尔福特菌和表皮葡萄球菌,其中大肠杆菌为肠道中常见条件致病菌,而SPN组胰腺的优势菌群为粘质沙雷菌、罗尔斯顿菌、戴尔福特菌、莱迪氏鞘氨醇单胞菌和柄杆菌。IPN组胰腺与肠道菌群共有ASV数105个,约占胰腺ASV数目29.2%,SPN组胰腺与肠道菌群共有ASV数10个,约占胰腺ASV数目10.3%。与SPN组相比,IPN组肠道和胰腺菌群更相似(P<0.001)。基于bray-curtis距离的β多样性距离比较同样表明,IPN组的肠道和胰腺菌群之间的距离比SPN组更接近(P<0.01)。4、IPN组与SPN组的肠道-血液菌群相关性:对IPN组和SPN组的血液菌群进行物种组成分析,在门水平,两组血液菌群均主要以变形菌门为主。在种水平,IPN组血液的优势菌群为罗尔斯顿菌、戴尔福特菌、莱迪氏鞘氨醇单胞菌、柄杆菌、大肠杆菌和屎肠球菌,其中大肠杆菌和屎肠球菌为肠道中常见条件致病菌,而SPN组血液的优势菌群为罗尔斯顿菌、戴尔福特菌、莱迪氏鞘氨醇单胞菌、柄杆菌和普通拟杆菌,其中普通拟杆菌为肠道中常见细菌。IPN组血液与肠道菌群共有ASV数145个,约占血液ASV数目21.7%,SPN组血液与肠道菌群共有ASV数42个,约占血液ASV数目10.1%。与SPN组相比,IPN组肠道和血液菌群更相似(P<0.001)。但是,IPN组和SPN组的肠道和血液菌群之间的β多样性距离无统计学差异(P>0.05)。5、IPN组与SPN组的肠道-肺部菌群相关性:对IPN组和SPN组的肺部菌群进行物种组成分析,在门水平,两组肺部菌群均以变形菌门、厚壁菌门和拟杆菌门为主。种水平上,IPN组肺部菌群的优势菌群为未分类的普雷沃氏菌、未分类的链球菌、铜绿假单胞菌、粘质沙雷菌和鲍曼不动杆菌,SPN组肺部菌群相对丰度较高的菌群为粘质沙雷菌、具核梭杆菌、未分类的链球菌、未分类的普雷沃氏菌和缓症链球菌,两组肺部的优势菌群均与肠道的优势菌群明显不同。然而,IPN组肺部与肠道菌群共有ASV数70个,约占肺部ASV数目4.3%,而SPN组肺部与肠道菌群共有ASV数13个,约占肺部ASV数目1.3%。与SPN组相比,IPN组肠道和肺部菌群更相似(P<0.001)。基于bray-curtis距离的β多样性距离比较同样表明,IPN组的肠道和肺部菌群之间的距离比SPN组更接近(P<0.05)。6、IPN组各部位菌群与肠道菌群的相关性差异:将IPN组患者各部位与肠道共有的ASV数目进行比较,IPN组胰腺菌群和肠道菌群的相似性最高(29.2%),其次是血液菌群和肠道菌群的相似性(21.7%),肺部菌群和肠道菌群的相似性最低(4.3%),两两比较的差异均有统计学意义(P<0.01)。结论:1、IPN患者和SPN患者的肠道菌群结构存在显著差异,主要体现在IPN患者的肠道菌群失衡(有益菌减少和致病菌增多),说明其可能在AP患者胰腺感染的发生发展中有着重要作用。2、IPN患者胰腺、血液、肺部的菌群与肠道菌群的同源性均较SPN患者更高,且IPN患者的胰腺菌群与肠道菌群最为相似,其次是血液菌群,肺部菌群与肠道菌群的相似性最低。这提示IPN患者各部位的感染可能部分来源于肠道,尤其是胰腺感染。更多还原

【Abstract】 Background And Aim:Acute pancreatitis(AP)is a common disease of digestive system,of which about20% develop into severe acute pancreatitis(SAP).Patients with SAP are prone to organ dysfunction in the early stage of the disease,and the mortality rate can reach15-20%,and if infectious complications occur in the later course of the disease,the mortality rate is as high as 30%.Intestinal-derived infection is considered to be the main cause of infectious complications in patients with AP,and relevant studies have detected intestinal conditional pathogenic bacteria in pancreatic necrotic drainage fluid and peripheral blood of AP patients,suggesting that they may enter the circulatory system and abdominal tissues and organs through the damaged intestinal mucosal barrier,causing intestinal-derived infection.However,no study has been conducted to directly determine the correlation between the flora of different sites and intestinal flora in patients with AP by retaining samples of pancreatic necrotic drainage fluid,peripheral blood and deep sputum.Based on this,the aim of this study was to analyze the flora of pancreatic necrotic drainage fluid,blood and deep sputum from patients with AP undergoing the first minimally invasive intervention for pancreatic necrosis,to analyze their homology with intestinal flora,and to further validate the theory of intestinal-derived infection in AP.Methods:This study prospectively included AP patients were hospitalized in the Department of Gastroenterology,the First Affiliated Hospital of Nanchang University,from October 2021 to March 2022.During the first minimally invasive intervention of pancreatic necrosis,samples of pancreatic necrotic drainage fluid,blood,fecal and deep sputum were collected at the same time.Based on Pac Bio SMRT sequencing technology,full-length 16 S r DNA sequencing was performed to analyze the structural characteristics of the pancreas,blood,intestinal and lungs of the patients.The clinical data of patients were collected.According to the results of laboratory culture of pancreatic necrotic drainage fluid during minimally invasive intervention of pancreatic necrosis,the patients were divided into infected pancreatic necrosis(IPN)group and sterile pancreatic necrosis(SPN)group.The difference of intestinal flora between the two groups and the correlation between pancreatic,blood,pulmonary flora and intestinal flora were compared between the two groups.Results:1.A total of 22 patients were included in this study,including 16 patients in the IPN group and 6 patients in the SPN group.There were no statistical differences in baseline data between the two groups in terms of gender,age,cause of AP,mode of first minimally invasive intervention,time from onset to admission and time from onset to first minimally invasive intervention.2.Composition characteristics and comparison of intestinal flora between IPN group and SPN group: α diversity showed that there was a trend of lower intestinal flora species diversity indices such as Chao1 index and Shannon index in IPN group than SPN group,but the difference was not statistically significant(P > 0.05).PCo A analysis based on bray-curtis distance showed that there was a significant difference in the structure of intestinal flora between the IPN and SPN groups(Adonis test,P <0.05).Further species composition analysis was performed,and the results showed that at the phylum level,the relative abundance of the beneficial phylum Verrucomicrobia was significantly reduced in the IPN group compared to the SPN group(0.9% vs.12.6%;P < 0.05);at the species level,the conditionally pathogenic bacteria Escherichia coli,Ralstonia sp,Sphingomonas leidyi and Caulobacter sp.were significantly more abundant in relative abundance in the IPN group compared to the SPN group(P < 0.05).The expression abundance of Escherichia coli in the fecal samples of both groups was further verified by q PCR,and the results showed that the relative abundance of Escherichia coli in the fecal of IPN group was significantly higher than that in the SPN group(P < 0.05).3.Intestinal-pancreatic flora correlation between IPN and SPN groups: Species composition analysis of the pancreatic flora of IPN and SPN groups,at the phylum level,the pancreatic flora of both groups was predominantly Proteobacteria.At the species level,the dominant pancreatic flora in the IPN group were Serratia marcescens、Ralstonia sp.、Escherichia coli、Delftia tsuruhatensis and Staphylococcus epidermidis,among them,Escherichia coli was the common conditionally pathogenic bacteria in the intestine,while the dominant flora in the pancreas of the SPN group were Serratia marcescens、Ralstonia sp.、Delftia tsuruhatensis、Sphingomonas leidyi、and Caulobacter sp..There were 105 ASVs in the pancreatic and intestinal flora in the IPN group,accounting for 29.2% of the number of ASVs in the pancreas,and 10 ASVs in the pancreatic and intestinal flora in the SPN group,accounting for 10.3% of the number of ASVs in the pancreas.The intestinal and pancreatic flora were more similar in the IPN group compared to the SPN group(P<0.001).Comparison of beta diversity distances based on bray-curtis distances likewise showed that the distances between intestinal and pancreatic flora were closer in the IPN group than in the SPN group(P<0.01).4.Intestinal-blood flora correlation between IPN and SPN groups: Species composition analysis of blood flora in IPN and SPN groups showed that at the phylum level,blood flora in both groups were predominantly Proteobacteria.At the species level,the dominant blood flora of the IPN group were Ralstonia sp.、Delftia tsuruhatensis 、 Sphingomonas leidyi 、 Caulobacter sp.、 Escherichia coli and Enterococcus faecium,where Escherichia coli、Enterococcus faecium were common conditionally pathogenic bacteria in the intestine,while the dominant groups of blood in the SPN group were Ralstonia sp.、Delftia tsuruhatensis、Sphingomonas leidyi、Caulobacter sp.and Bacteroides vulgatus,of which Bacteroides vulgatus was common in the intestine.The blood and intestinal flora in the IPN group had a total of145 ASVs,accounting for 21.7% of the blood ASVs,and the blood and intestinal flora in the SPN group had a total of 42 ASVs,accounting for 10.1% of the blood ASVs.The intestinal and blood flora were more similar in the IPN group compared with the SPN group(P<0.001).However,there was no statistical difference in the distance of beta diversity between the intestinal and blood flora of the IPN and SPN groups(P > 0.05).5.Intestinal-pulmonary flora correlation between IPN and SPN groups: Species composition analysis of pulmonary flora in IPN and SPN groups,at the phylum level,the pulmonary flora of both groups was dominated by Proteobacteria、Firmicutes、Bacteroidetes.At the species level,the dominant pulmonary flora in the IPN group were Prevotella＿＿unclassified 、 Streptococcus＿＿unclassified 、 Pseudomonas aeruginosa、Serratia marcescens and Acinetobacter baumannii,the dominant flora of the pulmonary flora of the SPN group were Serratia marcescens、Fusobacterium nucleatum、Streptococcus＿＿unclassified、Prevotella＿＿unclassified and Streptococcus mitis,and the dominant pulmonary flora of both groups were significantly different from the dominant flora of the intestine.However,the pulmonary and intestinal flora in the IPN group had a total of 70 ASVs,accounting for 4.3% of the pulmonary ASVs,whereas the pulmonary and intestinal flora in the SPN group had a total of 13 ASVs,accounting for 1.3% of the pulmonary ASVs.The intestinal and pulmonary flora were more similar in the IPN group compared to the SPN group(P<0.001).Comparison of beta diversity distances based on bray-curtis distances likewise showed that the distances between intestinal and pulmonary flora were closer in the IPN group than in the SPN group(P<0.05).6.The correlation difference between the flora of each part of the IPN group and the intestinal flora: Comparing the number of ASVs shared by each part of the patients in the IPN group with the intestinal flora,the similarity between the pancreatic flora and the intestinal flora in the IPN group was the highest(29.2%),followed by the similarity between the blood flora and the intestinal flora(21.7%),and the similarity between the pulmonary flora and the intestinal flora was the lowest(4.3%).The differences between the two comparisons were statistically significant(P< 0.01).Conclusions:(1)There are significant differences in the structure of intestinal flora between IPN patients and SPN patients,mainly in the imbalance of intestinal flora(decrease of beneficial bacteria and increase of pathogenic bacteria)in IPN patients,indicating that it may have an important role in the development of pancreatic infection in AP patients.(2)The homology of pancreatic,blood and pulmonary flora with intestinal flora is higher in IPN patients than in SPN patients,and the pancreatic flora of IPN patients is most similar to the intestinal flora,followed by the blood flora,and the pulmonary flora have the lowest similarity to the intestinal flora.This suggests that infections at various sites in IPN patients may be partially of intestinal origin,especially pancreatic infection.更多还原