肠道微生态在重症急性胰腺炎合并肠功能障碍中的作用研究

发布时间：2018-07-26 20:22

【摘要】：随着我国人民生活水平的提高和生活习惯的改变,AP的发病率日益升高。尽管在过去的几十年里,对于AP的诊治取得了重大进步,但SAP的死亡率依旧居高不下,仍在20-30%左右[1]。而SAP的发病原因复杂、可累及全身多个脏器的特点又加大了其治疗和研究的难度。根据大量研究结果显示,SAP常发生急性胃肠道损伤,造成肠道黏膜屏障功能障碍。肠道屏障主要由机械屏障、化学屏障、生物屏障和免疫屏障组成,正常情况下发挥阻隔肠道菌群及其有害产物进入血流和腹腔脏器的作用。当肠道屏障损伤时,肠黏膜通透性增加,肠道菌群便可穿过肠道屏障进入循环系统和腹腔组织器官,造成肠源性感染,这也是SAP患者后期死亡的主要原因。关于AP发生时肠道黏膜屏障功能障碍的发生机制有多种解释,但目前尚不完全清楚。Van等[2]人的动物实验也发现,在SAP条件下,肠道内存在革兰阳性球菌、革兰阴性杆菌和厌氧菌过度生长现象。这说明SAP中可能出现肠道菌群紊乱,破坏了原本稳定的微生物屏障结构,并影响到肠道屏障功能,促进了肠道菌群移位。若细菌产生的内毒素等产物入血,可刺激炎症细胞因子大量释放,加重全身炎症反应,并对胰腺造成“二次打击”,甚至引发或加重多器官功能衰竭,导致SAP患者死亡风险升高[3]。综上所述,我们推测肠道菌群变化可能参与了急性胰腺炎中的肠道黏膜屏障功能损伤和炎症反应,并对AP患者继发感染产生影响。为了验证上述研究假设,本研究特开展如下试验。一、目的检测AP患者肠道菌群的变化,研究肠道菌群对急性胰腺炎中肠道黏膜屏障和炎症反应的作用,以及肠道菌群对AP预后的影响。二、方法根据不同病情严重程度,对AP患者进行分组,主要分为重症急性胰腺炎组(SAP组,n=25)和轻症急性胰腺炎组(MAP组,n=37);重症急性胰腺炎组又可进一步细分为合并脏器衰竭的重症急性胰腺炎组(TSAP组,n=6)和中度重症急性胰腺炎组(MSAP组,n=19)。收集上述AP各个分组和健康人H组(n=31)的粪便、血清标本。对粪便标本细菌的16S r DNA V3-V4区采用高通量测序技术,检测肠道菌群多样性和丰度;对血清标本利用ELISA方法检测CRP、PCT和IL-6水平。此外,还收集急性胰腺炎患者临床资料,对病情进行评估,包括APACHE II评分和急性胃肠损伤(acute gastrointestinal injury,AGI)评分,以及随访患者有无继发感染等情况,对比不同分组检测结果,研究肠道菌群变化对炎症反应和肠道黏膜屏障的影响,以及肠道菌群对预后的影响。统计数据一律采用的是SPSS 18.0软件进行分析,其中将计量资料用均数±标准差(?x±SD)表示,而对组间比较采用的是t检验、单因素方差分析或非参数检验,相关性分析采用简单线性相关分析;分类资料用例数和百分比表示,组间比较采用Fishier确切概率检验或卡方检验;多因素分析采用logistic回归分析。三、结果(一)各组的CRP、PCT、IL-6水平和AGI分级比较1、各组的CRP、PCT、IL-6水平比较SAP组平均CRP水平为203.77±112.71 mg/L,PCT用中位数和四分位数表示为1.23(0.63,3.29)pm/m L,平均IL-6水平为74.25±78.04 pg/ml;MAP组平均CRP水平为103.50±62.14 mg/L,PCT水平为0.62(0.45,1.10)pm/m L,平均IL-6水平为32.15±27.88 pg/ml;H组平均CRP水平为5.88±3.86 mg/L,PCT水平为0.16(0.08,0.32)pm/m L,平均IL-6水平为4.89±3.52 pg/ml。根据单因素分析和t检验结果显示,和H组比较,SAP组和MAP组的CRP、PCT、IL-6水平明显升高,并且SAP组的CRP、PCT、IL-6水平升高比MAP组更为显著(P0.05)。TSAP组的CRP水平为217.67±67.37 mg/L,平均PCT水平为8.05±9.87 pm/m L,平均IL-6水平为63.90±23.88 pg/ml;MSAP组的CRP水平为199.38±124.87 mg/L,平均PCT水平为1.31±1.25 pm/m L,平均IL-6水平为77.52±88.96 pg/ml。根据t检验结果显示,TSAP组与MSAP组的CRP和IL-6水平接近,差异无统计学意义(P=0.737和P=0.718),而TSAP组的PCT水平显著升高,差异有统计学意义(P0.001)。2、各组的AGI分级比较SAP组和MAP组两组患者全部发生肠功能障碍,AGI分级采用住院7天内最高的AGI分级。SAP组最低AGI分级最高为Ⅳ级,最低为II级,其中AGIⅣ级的为4%(n=1),AGIⅢ级的为20%(n=5),AGIⅡ级的为76%(n=19);MAP组AGI分级最高为Ⅱ级,最低为I级,其中AGIⅡ级为10.8%(n=4),AGIⅠ级为89.2%(n=33)。而H组的健康对照人群无肠功能障碍,AGI分级100%为0级(n=31)。根据Fisher确切概率检验,三组AGI分级差异有统计学意义,并且SAP组的AGI分级明显高于MAP组的AGI分级(P0.001)。TSAP组AGIⅣ级的为16.7%(n=1),AGIⅢ级的为83.3%(n=5);而MSAP组AGIⅢ级的为100%(n=19)。根据Fisher确切概率检验结果显示,TSAP组和MSAP组的AGI分级差异有统计学意义,并且TSAP组的AGI分级要比MSAP组的高(P0.001)。(二)急性胰腺炎中的肠道菌群变化分析根据单因素分析和t检验结果显示,SAP组、MAP组和H组的肠道细菌总量一致,差异没有统计学意义(P0.05);但和H组比较,SAP组和MAP组肠道菌群丰度发生显著变化;而与MSAP组相比,TSAP组的肠道菌群也发生了部分优势菌的丰度变化。1、菌群多样性的比较根据Alpha多样性分析,SAP组、MAP组和H组的丰富度指数observed species指数、chao1指数和PD_whole_tree指数、多样性指数shannon指数和simpson指数并无显著差异(P0.05),说明3组间的物种多样性无显著差异。2、菌群丰度的比较根据非参数检验结果显示,AP患者的肠道菌群丰度在各个分类水平上均发生了显著变化。在门水平上,拟杆菌门丰度下降、变形菌门丰度升高;在纲水平上,拟杆菌纲丰度下降、丙型变形菌纲丰度升高;在目水平上,拟杆菌目丰度下降、肠杆菌目丰度升高;在科水平上,拟杆菌科和毛螺菌科丰度下降,而肠杆菌科和肠球菌科丰度升高;在属水平上,拟杆菌属、罗氏菌属、萨特氏菌属和考拉杆菌属丰度明显下降,埃希菌属/志贺菌属、肠球菌属和乳酸杆菌属丰度明显升高。和MSAP组相比,TSAP组的埃希菌属/志贺菌属和萨特氏菌属丰度显著下降,差异具有统计学意义。(三)肠道菌群在急性胰腺炎中对炎症反应和肠道黏膜屏障的作用将肠道优势菌丰度与CRP、PCT和IL-6水平进行简单线性相关分析,结果显示不同分类水平的肠道菌群丰度与CRP、PCT和IL-6水平具有显著相关性。AP条件下,CRP、PCT和IL-6水平的升高和组间差异的形成,与拟杆菌、毛螺菌、罗氏菌、萨特氏菌和考拉杆菌等肠道细菌出现对应各个分类的丰度下降以及变形菌、肠杆菌、肠球菌在各个分类水平的丰度升高有关。综合以上研究结果,可知多种肠道菌群可能参与了AP中的炎症反应,并在其中起了抑制或促进炎症反应的不同作用。根据简单线性相关分析结果显示,不同分类水平的肠道菌群丰度与AGI分级具有相关关系。其中,拟杆菌、毛螺菌、罗氏菌、分类位置未定的毛螺菌、普氏菌和梭状菌等肠道细菌可能对肠黏膜具有保护作用,而变性杆菌、肠杆菌、埃希菌/志贺菌、假单胞菌等肠道细菌可能对肠黏膜具有损伤作用。这些肠道菌群极有可能在AP中参与肠功能障碍的发生和发展,并发挥着不同的作用。(四)肠道菌群和CRP、PCT、IL-6、AGI分级在预测SAP继发感染中的作用SAP组25人中共有6人发生胰腺坏死组织感染,其中TSAP组有5人发生感染,MSAP组有1人感染。对SAP组患者是否继发感染的可能影响因素进行logistic回归分析,这些因素包括:CRP、PCT和IL-6水平、AGI分级以及上述在不同研究对象分组中具有差异(P0.05)、与炎症指标或AGI分级相关的优势菌等8个因素。结果显示只有PCT和AGI分级对SAP患者继发感染的影响具有统计学意义,AP分级越高,SAP患者继发感染的风险越高;而CRP、IL-6和肠道菌群对于SAP患者继发感染没有显著影响。四、结论肠道菌群变化可能参与了AP中的肠黏膜损伤和炎症反应,并与病情严重程度有关,但其优势菌的丰度水平与SAP患者继发感染的风险可能没有明显相关关系。
[Abstract]:With the improvement of people's living standard and the change of living habits, the incidence of AP is increasing. Although significant progress has been made in the diagnosis and treatment of AP in the past few decades, the mortality of SAP is still high, and the cause of SAP is still at 20-30% [1]. and the cause of the disease is complex. The difficulty of treatment and research. According to the results of a large number of studies, SAP often causes acute gastrointestinal damage and causes intestinal mucosal barrier dysfunction. The intestinal barrier is composed mainly of mechanical barriers, chemical barriers, biological barriers and immune barriers. In normal cases, intestinal barrier and its harmful products are used to enter the blood flow and abdominal organs. When the intestinal barrier is damaged, intestinal mucosal permeability increases, intestinal microflora can pass through the intestinal barrier into the circulatory system and intraperitoneal tissue, causing intestinal infection, which is also the main cause of the late death of SAP patients. There are many explanations about the mechanism of intestinal mucosal barrier dysfunction at the time of AP, but it is not complete at present. .Van and other [2] animal experiments also found that in the SAP condition, there are gram positive cocci, gram-negative bacilli and anaerobes in the intestinal tract. This indicates that the intestinal flora disorder may occur in the SAP, which destroys the original stable microbial barrier structure, and affects the intestinal barrier function and promotes the intestinal microflora shift. The entry of endotoxin produced by bacteria into blood can stimulate the release of inflammatory cytokines, aggravate the systemic inflammatory response, and cause "two strikes" to the pancreas, even cause or aggravate multiple organ failure, which leads to the increase of the risk of death in SAP patients [3].. We speculate that intestinal flora changes may be involved in acute pancreatitis. The effects of intestinal mucosal barrier function damage and inflammatory response on the secondary infection of AP patients. In order to verify the hypothesis mentioned above, the following experiments were carried out in this study. 1. Objective to detect the changes in intestinal flora in patients with AP and to study the effect of intestinal flora on intestinal mucosal barrier and inflammatory response in acute pancreatitis, and intestinal microflora The effect of AP prognosis. Two, according to the severity of the disease, the patients were divided into groups of AP, which were divided into severe acute pancreatitis group (group SAP, n=25) and mild acute pancreatitis (group MAP, n=37); severe acute pancreatitis could be further subdivided into severe acute pancreatitis (group TSAP, n=6) and moderate severe acute pancreatitis (TSAP group, n=6). Sexual pancreatitis group (group MSAP, n=19). The faeces and serum specimens of each group of AP and healthy people H (n=31) were collected. The diversity and abundance of intestinal flora were detected by high throughput sequencing technology in the 16S R DNA V3-V4 region of fecal specimens. The serum specimens were detected by ELISA square method and CRP, PCT, and levels were detected. In addition, the acute pancreatitis was also collected. The patient's clinical data were evaluated, including the APACHE II score and the acute gastrointestinal injury (acute gastrointestinal injury, AGI) scores, and the follow-up of patients with secondary infection. The effects of intestinal microflora changes on the inflammatory response and intestinal mucosal barrier and the prognosis of intestinal microflora were compared. All the statistical data are analyzed with SPSS 18 software, which are measured with mean number + standard deviation (? X + SD), while the comparison between groups is t test, single factor variance analysis or non parametric test, and correlation analysis using simple linear correlation analysis; classification data use case number and percentage representation, inter group ratio Fishier accurate probability test or chi square test was used. Logistic regression analysis was used in multifactor analysis. Three. Results (1) the CRP, PCT, IL-6 level and AGI classification of each group were compared to 1. The average CRP level of CRP, PCT and IL-6 in each group was 203.77 + 112.71 mg/L, and the median and four quantiles were 1.23. The level of L-6 was 74.25 + 78.04 pg/ml, and the average CRP level in group MAP was 103.50 + 62.14 mg/L, PCT level was 0.62 (0.45,1.10) pm/m L, and the average IL-6 level was 32.15 + 27.88 pg/ml, and the average level of H group was 5.88 + 3.86, and the level was 0.16. The average level was 4.89 + 3.52. Compared with the H group, the level of CRP, PCT and IL-6 in group SAP and MAP was significantly higher, and the level of CRP, PCT, IL-6 in SAP group was more significant than that of MAP group (P0.05) was 217.67 + 67.37, the average level was 8.05 + 9.87, and the average level was 63.90 + 23.88. The average level of the group was 199.38 + 124.87. The level was 1.31 + 1.25 pm/m L, the average IL-6 level was 77.52 + 88.96 pg/ml., according to the t test results, the TSAP group was close to the CRP and IL-6 levels in the MSAP group. The difference was not statistically significant (P=0.737 and P=0.718), but the level of the TSAP group was significantly higher, the difference was statistically significant. All intestinal dysfunction occurred. The lowest level of AGI in group AGI of the highest AGI grade.SAP group in 7 days of hospitalization was grade IV, the lowest was grade II, of which AGI IV was 4% (n=1), AGI III was 20% (n=5), AGI II was 76% (n=19), and the lowest grade of MAP group was grade II, and the grade II was 10.8% 33) and the healthy control group of the H group had no intestinal dysfunction, and the AGI grade 100% was 0 (n=31). According to the exact probability test of Fisher, the difference in the AGI classification of the three groups was statistically significant, and the AGI classification of the SAP group was significantly higher than that of the AGI grade (P0.001).TSAP group of the MAP group (P0.001).TSAP group (16.7%), and the grade III of the class III was 100. % (n=19). According to the exact Fisher test results, the AGI classification difference between group TSAP and MSAP group was statistically significant, and the AGI classification of group TSAP was higher than that of MSAP group (P0.001). (two) the analysis of intestinal microflora in acute pancreatitis showed that the total amount of intestinal bacteria in SAP group, MAP group and group were the same according to the results of single factor analysis and t test. The difference was not statistically significant (P0.05), but compared with the H group, the abundance of intestinal flora in the SAP group and the MAP group changed significantly. Compared with the MSAP group, the intestinal microflora of the TSAP group also changed the abundance of some dominant bacteria, and the diversity of the flora was compared according to the Alpha diversity, and the richness index of the SAP group, the MAP group and the H group was similar to that of the Alpha diversity. The index, Chao1 index and PD_whole_tree index, diversity index Shannon index and Simpson index did not differ significantly (P0.05), indicating that there was no significant difference in species diversity between the 3 groups, and the comparison of the abundance of the flora showed that the intestinal microflora of the AP patients had significantly changed at the different levels. At the level, the abundance of bacteriobacteria decreased and the abundance of P. C. increased; at the level of the order, the abundance of bacilli decreased and the abundance of Enterobacteriaceae increased; at the level of the family, the abundances of the Bacillaceae and the family clonaceae decreased and the abundance of Enterobacteriaceae and enterococcaceae increased. At the level of the genus, the abundances of the genus and the genus salibacillum significantly decreased, and the abundance of the genus and Shigella, enterococci and lactobacilli significantly increased. Compared with the MSAP group, the abundance of the genus and Shigella and Salmonella in the TSAP group decreased significantly. (three) the intestinal tract was statistically significant. The effect of bacterial flora on inflammation and intestinal mucosal barrier in acute pancreatitis was a simple linear correlation analysis of the abundance of intestinal predominant bacteria and the levels of CRP, PCT and IL-6. The results showed that the abundance of intestinal flora at different levels of intestinal flora was significantly correlated with the levels of CRP, PCT and IL-6 in.AP conditions, the level of CRP, PCT and IL-6, and the difference between groups. The formation of intestinal bacteria, such as bacteriobacteria, spiralis, Roche, Salmonella and bacilli, was associated with the decrease in abundance and the increase in the abundance of deformable bacteria, enterobacteria and Enterococcus in various classification levels. According to the results of simple linear correlation analysis, the abundances of intestinal flora of different classification levels were related to the AGI classification. Among them, the intestinal bacteria such as bacilli, hairy snails, rosiella, undetermined Taxus, Prunus and Clostridium could protect the intestinal mucosa. Enterobacteriaceae, Enterobacteriaceae, Escherichia, Shigella, Shigella, Pseudomonas and other intestinal bacteria may have damage to intestinal mucosa. These intestinal flora are likely to participate in the occurrence and development of intestinal dysfunction in AP and play different roles. (four) intestinal microflora and CRP, PCT, IL-6, AGI classification in the prediction of secondary infection of SAP 6 of the 25 people in group SAP were infected with pancreatic necrosis, of which 5 were infected in group TSAP and 1 in group MSAP were infected. Logistic regression analysis on the possible influence factors of secondary infection in group SAP, including CRP, PCT and IL-6 levels, AGI classification, and the differences in the groups of different subjects (P0) .05), 8 factors such as the inflammatory markers or the dominant bacteria associated with AGI classification. The results showed that only PCT and AGI grading had a statistically significant effect on secondary infection in SAP patients, the higher the AP grade, the higher the risk of secondary infection in SAP patients; CRP, IL-6 and intestinal microflora had no significant impact on secondary infection in SAP patients. Four, the conclusion of intestinal microflora The changes may be involved in intestinal mucosal injury and inflammatory response in AP, and are related to the severity of the disease, but the abundance of the dominant bacteria may not be significantly related to the risk of secondary infection in SAP patients.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R576

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