【摘要】：隨著我國(guó)人民生活水平的提高和生活習(xí)慣的改變,AP的發(fā)病率日益升高。盡管在過(guò)去的幾十年里,對(duì)于AP的診治取得了重大進(jìn)步,但SAP的死亡率依舊居高不下,仍在20-30%左右[1]。而SAP的發(fā)病原因復(fù)雜、可累及全身多個(gè)臟器的特點(diǎn)又加大了其治療和研究的難度。根據(jù)大量研究結(jié)果顯示,SAP常發(fā)生急性胃腸道損傷,造成腸道黏膜屏障功能障礙。腸道屏障主要由機(jī)械屏障、化學(xué)屏障、生物屏障和免疫屏障組成,正常情況下發(fā)揮阻隔腸道菌群及其有害產(chǎn)物進(jìn)入血流和腹腔臟器的作用。當(dāng)腸道屏障損傷時(shí),腸黏膜通透性增加,腸道菌群便可穿過(guò)腸道屏障進(jìn)入循環(huán)系統(tǒng)和腹腔組織器官,造成腸源性感染,這也是SAP患者后期死亡的主要原因。關(guān)于AP發(fā)生時(shí)腸道黏膜屏障功能障礙的發(fā)生機(jī)制有多種解釋,但目前尚不完全清楚。Van等[2]人的動(dòng)物實(shí)驗(yàn)也發(fā)現(xiàn),在SAP條件下,腸道內(nèi)存在革蘭陽(yáng)性球菌、革蘭陰性桿菌和厭氧菌過(guò)度生長(zhǎng)現(xiàn)象。這說(shuō)明SAP中可能出現(xiàn)腸道菌群紊亂,破壞了原本穩(wěn)定的微生物屏障結(jié)構(gòu),并影響到腸道屏障功能,促進(jìn)了腸道菌群移位。若細(xì)菌產(chǎn)生的內(nèi)毒素等產(chǎn)物入血,可刺激炎癥細(xì)胞因子大量釋放,加重全身炎癥反應(yīng),并對(duì)胰腺造成“二次打擊”,甚至引發(fā)或加重多器官功能衰竭,導(dǎo)致SAP患者死亡風(fēng)險(xiǎn)升高[3]。綜上所述,我們推測(cè)腸道菌群變化可能參與了急性胰腺炎中的腸道黏膜屏障功能損傷和炎癥反應(yīng),并對(duì)AP患者繼發(fā)感染產(chǎn)生影響。為了驗(yàn)證上述研究假設(shè),本研究特開(kāi)展如下試驗(yàn)。一、目的檢測(cè)AP患者腸道菌群的變化,研究腸道菌群對(duì)急性胰腺炎中腸道黏膜屏障和炎癥反應(yīng)的作用,以及腸道菌群對(duì)AP預(yù)后的影響。二、方法根據(jù)不同病情嚴(yán)重程度,對(duì)AP患者進(jìn)行分組,主要分為重癥急性胰腺炎組(SAP組,n=25)和輕癥急性胰腺炎組(MAP組,n=37);重癥急性胰腺炎組又可進(jìn)一步細(xì)分為合并臟器衰竭的重癥急性胰腺炎組(TSAP組,n=6)和中度重癥急性胰腺炎組(MSAP組,n=19)。收集上述AP各個(gè)分組和健康人H組(n=31)的糞便、血清標(biāo)本。對(duì)糞便標(biāo)本細(xì)菌的16S r DNA V3-V4區(qū)采用高通量測(cè)序技術(shù),檢測(cè)腸道菌群多樣性和豐度;對(duì)血清標(biāo)本利用ELISA方法檢測(cè)CRP、PCT和IL-6水平。此外,還收集急性胰腺炎患者臨床資料,對(duì)病情進(jìn)行評(píng)估,包括APACHE II評(píng)分和急性胃腸損傷(acute gastrointestinal injury,AGI)評(píng)分,以及隨訪患者有無(wú)繼發(fā)感染等情況,對(duì)比不同分組檢測(cè)結(jié)果,研究腸道菌群變化對(duì)炎癥反應(yīng)和腸道黏膜屏障的影響,以及腸道菌群對(duì)預(yù)后的影響。統(tǒng)計(jì)數(shù)據(jù)一律采用的是SPSS 18.0軟件進(jìn)行分析,其中將計(jì)量資料用均數(shù)±標(biāo)準(zhǔn)差(?x±SD)表示,而對(duì)組間比較采用的是t檢驗(yàn)、單因素方差分析或非參數(shù)檢驗(yàn),相關(guān)性分析采用簡(jiǎn)單線性相關(guān)分析;分類資料用例數(shù)和百分比表示,組間比較采用Fishier確切概率檢驗(yàn)或卡方檢驗(yàn);多因素分析采用logistic回歸分析。三、結(jié)果(一)各組的CRP、PCT、IL-6水平和AGI分級(jí)比較1、各組的CRP、PCT、IL-6水平比較SAP組平均CRP水平為203.77±112.71 mg/L,PCT用中位數(shù)和四分位數(shù)表示為1.23(0.63,3.29)pm/m L,平均IL-6水平為74.25±78.04 pg/ml;MAP組平均CRP水平為103.50±62.14 mg/L,PCT水平為0.62(0.45,1.10)pm/m L,平均IL-6水平為32.15±27.88 pg/ml;H組平均CRP水平為5.88±3.86 mg/L,PCT水平為0.16(0.08,0.32)pm/m L,平均IL-6水平為4.89±3.52 pg/ml。根據(jù)單因素分析和t檢驗(yàn)結(jié)果顯示,和H組比較,SAP組和MAP組的CRP、PCT、IL-6水平明顯升高,并且SAP組的CRP、PCT、IL-6水平升高比MAP組更為顯著(P0.05)。TSAP組的CRP水平為217.67±67.37 mg/L,平均PCT水平為8.05±9.87 pm/m L,平均IL-6水平為63.90±23.88 pg/ml;MSAP組的CRP水平為199.38±124.87 mg/L,平均PCT水平為1.31±1.25 pm/m L,平均IL-6水平為77.52±88.96 pg/ml。根據(jù)t檢驗(yàn)結(jié)果顯示,TSAP組與MSAP組的CRP和IL-6水平接近,差異無(wú)統(tǒng)計(jì)學(xué)意義(P=0.737和P=0.718),而TSAP組的PCT水平顯著升高,差異有統(tǒng)計(jì)學(xué)意義(P0.001)。2、各組的AGI分級(jí)比較SAP組和MAP組兩組患者全部發(fā)生腸功能障礙,AGI分級(jí)采用住院7天內(nèi)最高的AGI分級(jí)。SAP組最低AGI分級(jí)最高為Ⅳ級(jí),最低為II級(jí),其中AGIⅣ級(jí)的為4%(n=1),AGIⅢ級(jí)的為20%(n=5),AGIⅡ級(jí)的為76%(n=19);MAP組AGI分級(jí)最高為Ⅱ級(jí),最低為I級(jí),其中AGIⅡ級(jí)為10.8%(n=4),AGIⅠ級(jí)為89.2%(n=33)。而H組的健康對(duì)照人群無(wú)腸功能障礙,AGI分級(jí)100%為0級(jí)(n=31)。根據(jù)Fisher確切概率檢驗(yàn),三組AGI分級(jí)差異有統(tǒng)計(jì)學(xué)意義,并且SAP組的AGI分級(jí)明顯高于MAP組的AGI分級(jí)(P0.001)。TSAP組AGIⅣ級(jí)的為16.7%(n=1),AGIⅢ級(jí)的為83.3%(n=5);而MSAP組AGIⅢ級(jí)的為100%(n=19)。根據(jù)Fisher確切概率檢驗(yàn)結(jié)果顯示,TSAP組和MSAP組的AGI分級(jí)差異有統(tǒng)計(jì)學(xué)意義,并且TSAP組的AGI分級(jí)要比MSAP組的高(P0.001)。(二)急性胰腺炎中的腸道菌群變化分析根據(jù)單因素分析和t檢驗(yàn)結(jié)果顯示,SAP組、MAP組和H組的腸道細(xì)菌總量一致,差異沒(méi)有統(tǒng)計(jì)學(xué)意義(P0.05);但和H組比較,SAP組和MAP組腸道菌群豐度發(fā)生顯著變化;而與MSAP組相比,TSAP組的腸道菌群也發(fā)生了部分優(yōu)勢(shì)菌的豐度變化。1、菌群多樣性的比較根據(jù)Alpha多樣性分析,SAP組、MAP組和H組的豐富度指數(shù)observed species指數(shù)、chao1指數(shù)和PD_whole_tree指數(shù)、多樣性指數(shù)shannon指數(shù)和simpson指數(shù)并無(wú)顯著差異(P0.05),說(shuō)明3組間的物種多樣性無(wú)顯著差異。2、菌群豐度的比較根據(jù)非參數(shù)檢驗(yàn)結(jié)果顯示,AP患者的腸道菌群豐度在各個(gè)分類水平上均發(fā)生了顯著變化。在門水平上,擬桿菌門豐度下降、變形菌門豐度升高;在綱水平上,擬桿菌綱豐度下降、丙型變形菌綱豐度升高;在目水平上,擬桿菌目豐度下降、腸桿菌目豐度升高;在科水平上,擬桿菌科和毛螺菌科豐度下降,而腸桿菌科和腸球菌科豐度升高;在屬水平上,擬桿菌屬、羅氏菌屬、薩特氏菌屬和考拉桿菌屬豐度明顯下降,埃希菌屬/志賀菌屬、腸球菌屬和乳酸桿菌屬豐度明顯升高。和MSAP組相比,TSAP組的埃希菌屬/志賀菌屬和薩特氏菌屬豐度顯著下降,差異具有統(tǒng)計(jì)學(xué)意義。(三)腸道菌群在急性胰腺炎中對(duì)炎癥反應(yīng)和腸道黏膜屏障的作用將腸道優(yōu)勢(shì)菌豐度與CRP、PCT和IL-6水平進(jìn)行簡(jiǎn)單線性相關(guān)分析,結(jié)果顯示不同分類水平的腸道菌群豐度與CRP、PCT和IL-6水平具有顯著相關(guān)性。AP條件下,CRP、PCT和IL-6水平的升高和組間差異的形成,與擬桿菌、毛螺菌、羅氏菌、薩特氏菌和考拉桿菌等腸道細(xì)菌出現(xiàn)對(duì)應(yīng)各個(gè)分類的豐度下降以及變形菌、腸桿菌、腸球菌在各個(gè)分類水平的豐度升高有關(guān)。綜合以上研究結(jié)果,可知多種腸道菌群可能參與了AP中的炎癥反應(yīng),并在其中起了抑制或促進(jìn)炎癥反應(yīng)的不同作用。根據(jù)簡(jiǎn)單線性相關(guān)分析結(jié)果顯示,不同分類水平的腸道菌群豐度與AGI分級(jí)具有相關(guān)關(guān)系。其中,擬桿菌、毛螺菌、羅氏菌、分類位置未定的毛螺菌、普氏菌和梭狀菌等腸道細(xì)菌可能對(duì)腸黏膜具有保護(hù)作用,而變性桿菌、腸桿菌、埃希菌/志賀菌、假單胞菌等腸道細(xì)菌可能對(duì)腸黏膜具有損傷作用。這些腸道菌群極有可能在AP中參與腸功能障礙的發(fā)生和發(fā)展,并發(fā)揮著不同的作用。(四)腸道菌群和CRP、PCT、IL-6、AGI分級(jí)在預(yù)測(cè)SAP繼發(fā)感染中的作用SAP組25人中共有6人發(fā)生胰腺壞死組織感染,其中TSAP組有5人發(fā)生感染,MSAP組有1人感染。對(duì)SAP組患者是否繼發(fā)感染的可能影響因素進(jìn)行l(wèi)ogistic回歸分析,這些因素包括:CRP、PCT和IL-6水平、AGI分級(jí)以及上述在不同研究對(duì)象分組中具有差異(P0.05)、與炎癥指標(biāo)或AGI分級(jí)相關(guān)的優(yōu)勢(shì)菌等8個(gè)因素。結(jié)果顯示只有PCT和AGI分級(jí)對(duì)SAP患者繼發(fā)感染的影響具有統(tǒng)計(jì)學(xué)意義,AP分級(jí)越高,SAP患者繼發(fā)感染的風(fēng)險(xiǎn)越高;而CRP、IL-6和腸道菌群對(duì)于SAP患者繼發(fā)感染沒(méi)有顯著影響。四、結(jié)論腸道菌群變化可能參與了AP中的腸黏膜損傷和炎癥反應(yīng),并與病情嚴(yán)重程度有關(guān),但其優(yōu)勢(shì)菌的豐度水平與SAP患者繼發(fā)感染的風(fēng)險(xiǎn)可能沒(méi)有明顯相關(guān)關(guān)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.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R576

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