本文選題：肝炎 + 乙型�。� 參考：《第三軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：慢性乙型肝炎病毒(HBV)感染是一個威脅全球健康的重要公共衛(wèi)生問題,我國慢性HBV感染者高達(dá)約9300萬,約有10%-30%的患者出現(xiàn)自發(fā)肝炎急性加重(acute exacerbation,AE),部分進(jìn)展為輕度肝炎,部分進(jìn)展為重度肝炎以及慢加急性肝衰竭(acute-on-chronic liver failure,ACLF)[1-2]。慢性HBV感染者一旦進(jìn)展為重型肝炎/ACLF,常合并消化道出血、自發(fā)性細(xì)菌性腹膜炎(spontaneous bacterial peritonitis,SBP)、肝性腦病等并發(fā)癥,死亡率極高[3],盡管生物人工肝等器官衰竭的支持治療已取得進(jìn)步,但仍缺乏特異的內(nèi)科治療手段。最近幾年,ACLF的發(fā)病機(jī)理研究取得諸多進(jìn)展,免疫功能的紊亂失衡、小腸細(xì)菌異常增生所致細(xì)菌易位以及微循環(huán)障礙被認(rèn)為是影響ACLF進(jìn)展的重要因素[4]。在肝硬化及重型肝炎時,腸道血供及免疫狀態(tài)受到明顯影響,腸道微環(huán)境改變,可能導(dǎo)致腸道微生態(tài)平衡狀態(tài)發(fā)生失衡。自發(fā)性細(xì)菌性腹膜炎是重型肝炎/慢加急肝衰竭患者的一種常見并發(fā)癥,是導(dǎo)致肝功能惡化的常見誘因之一,腸道微生態(tài)失衡及細(xì)菌易位被認(rèn)為是導(dǎo)致SBP發(fā)生的主要機(jī)制[5]。近年來,越來越多的研究也通過16S rDNA和宏基因組等技術(shù)揭示出腸道菌群在慢性肝臟疾病中的改變,以及腸道微生態(tài)失衡參與慢性肝臟疾病及其各種并發(fā)癥的發(fā)生發(fā)展。然而關(guān)于慢性乙型肝炎(chronic hepatitis B,CHB)疾病不同階段腸道菌群的變化仍不清,特別是關(guān)于乙肝相關(guān)慢加急性肝衰竭(HBV-ACLF)和CHB疾病不同嚴(yán)重程度的腸道菌群變化仍然不清楚。為了全面分析CHB患者腸道微生態(tài)失衡情況,探索HBV-ACLF的腸道菌群結(jié)構(gòu)變化特征,為理解腸道菌群在重型肝炎病理機(jī)制中的作用提供依據(jù)。第一部分HBV-ACLF患者糞便菌群結(jié)構(gòu)多樣性研究,以HBV-ACLF為研究對象,慢性乙型肝炎重度(severe chronic hepatitis B,S-CHB)、慢性乙型肝炎輕中度(mild and moderate chronic hepatitis B,M-CHB)及健康人群(healthy controls,HC)為對照組,提取糞便樣本基因組DNA,運(yùn)用基于16S rDNA的V3-V4區(qū)高通量測序方法分析研究對象的腸道菌群結(jié)構(gòu)、豐度及多樣性特點(diǎn),研究HBV-ACLF和CHB不同疾病階段腸道菌群的差異及變化特征。第二部分通過回顧性分析自發(fā)性細(xì)菌性腹膜炎(SBP)患者腹水培養(yǎng)陽性的微生物分布特點(diǎn)、通過16S rDNA高通量測序探索分析培養(yǎng)陰性腹水樣本細(xì)菌存在的可能性及種類,以期探索SBP感染與腸道菌群的關(guān)系,并評估感染標(biāo)志物降鈣素原(PCT)和血常規(guī)中性粒細(xì)胞在SBP中的變化及診斷效能。本研究主要結(jié)果如下:主要結(jié)果:1.重型乙型肝炎/HBV-ACLF患者腸道菌群16S rDNA高通量測序結(jié)果顯示:HBV-ACLF患者腸道菌群OTU數(shù)目、community richness指數(shù)顯著低于M-CHB患者和健康組,提示HBV-ACLF患者腸道菌群豐度顯著降低;NMDS分析顯示HBV-ACLF組可分別與M-CHB組、健康對照組清晰分開,提示HBV-ACLF患者腸道菌群整體結(jié)構(gòu)顯著不同于M-CHB和健康組,去除伴有肝硬化的患者后,NMDS分析仍可清晰區(qū)分,提示HBV-ACLF患者無論是否伴有肝硬化其腸道菌群結(jié)構(gòu)均發(fā)生顯著變化;2.HBV-ACLF患者腸道菌群發(fā)生廣泛的群落改變,采用M-CHB和HBV-ACLF患者測序得到的所有菌屬,通過隨機(jī)森林分析可顯著區(qū)分M-CHB和ACLF患者人群,且獲得屬水平的變量重要性排序。提示采用腸道菌群分類注釋的屬水平,可顯著區(qū)分兩種疾病狀態(tài)的患者,不同菌屬在區(qū)分兩組患者時其變量重要性不同;3.Veillonella,Streptococcus,Fusobacterium等22個屬在HBV-ACLF患者中顯著增高,其豐度升高的程度與TB、INR、MELD評分呈顯著正相關(guān);Romboutsia等26個屬顯著富集在M-CHB患者中,其豐度與TB、INR、MELD評分呈顯著負(fù)相關(guān),提示48個特征菌屬與評價疾病嚴(yán)重程度的臨床指標(biāo)具有相關(guān)性;4.腸道菌群特征菌屬的豐度在慢乙肝急性發(fā)作不同嚴(yán)重程度表型狀態(tài)下的演替過程顯示:M-CHB-富集菌屬(MEG)累積豐度作為潛在有益菌,ACLF-富集菌屬(AEG)累積豐度作為潛在致病菌,在HC組(MEG顯著高于AEG,P0.05),M-CHB組(MEG顯著高于AEG,P0.05),S-CHB組(MEG接近AEG,P0.05),ACLF組(MEG顯著低于AEG,P0.05);隨著疾病的加重MEG累積豐度顯著降低,AEG累積豐度顯著增高,ACLF患者中兩類菌屬豐度比例倒置;5.自發(fā)性細(xì)菌性腹膜炎特點(diǎn)分析發(fā)現(xiàn),SBP患者的腹水培養(yǎng)陽性率低,腹水培養(yǎng)常見細(xì)菌為大腸埃希氏菌、肺炎克雷伯菌、鏈球菌屬、葡萄球菌屬等;有腹膜刺激征的患者但培養(yǎng)陰性的腹水樣本經(jīng)16S rDNA高通量測序初步探索分析提示可能存在一種或幾種細(xì)菌或細(xì)菌DNA;腹水中檢出的細(xì)菌種類與HBV-ACLF患者腸道菌群中異常增高的潛在致病菌種類部分一致;6.通過ROC曲線分析評估感染指標(biāo)血降鈣素原(PCT)和血常規(guī)中性粒細(xì)胞比例在SBP患者中的診斷效能,結(jié)果顯示PCT在SBP1組(培養(yǎng)陽性PMN≥250×106/L),SBP2組(培養(yǎng)陽性PMN250×106/L),SBP3組(培養(yǎng)陰性PMN≥250×106/L)的最佳CUT-OFF值分別為:0.795 ng/m L、0.265 ng/m L、0.405 ng/m L;AUC值依次為:0.963、0.767、0.714;敏感度依次為:100.00%、90.00%、62.30%;特異度依次為:92.70%、63.40%、80.50%;血常規(guī)Neu%診斷的最佳界值分別為:68.45%、62.65%、65.00%;AUC值依次為:0.878、0.756、0.669。依據(jù)上述界值,降鈣素原和中性粒細(xì)胞比例兩者串聯(lián)診斷SBP1、SBP2、SBP3組的AUC值依次為:0.976、0.865、0.706。結(jié)論:本研究基于高通量測序方法首次分析并揭示了HBV-ACLF及CHB不同疾病進(jìn)展階段的腸道菌群紊亂特征,隨著CHB疾病嚴(yán)重程度的增加,腸道菌群呈現(xiàn)進(jìn)行性、結(jié)構(gòu)性紊亂,整體群落豐度顯著降低;HBV-ACLF患者腸道菌群顯著區(qū)別于M-CHB患者,其腸道菌群的平衡被打破,潛在致病菌和有益菌豐度比例倒置;HBV-ACLF患者腸道菌群中異常增高的潛在致病菌種類與SBP患者腹水中檢出的細(xì)菌種類部分一致;本研究提示異常增高的潛在致病菌可能增加感染風(fēng)險從而在ACLF發(fā)生、發(fā)展過程中扮演重要作用,為通過以腸道菌群為靶點(diǎn)進(jìn)行ACLF的診斷評估和治療提供數(shù)據(jù)支持。
[Abstract]:Chronic hepatitis B virus (HBV) infection is an important public health problem threatening global health. There are about 93 million of chronic HBV infection in China, with acute exacerbation of spontaneous hepatitis (acute exacerbation, AE) in some patients with 10%-30%, partial progression to mild hepatitis and partial progression to severe hepatitis and chronic acute liver failure (acute-on-). Chronic liver failure, ACLF) [1-2]. chronic HBV infected persons, once progressing to severe hepatitis /ACLF, often merge digestive tract bleeding, spontaneous bacterial peritonitis (spontaneous bacterial peritonitis, SBP), hepatic encephalopathy and so on, the mortality rate is very high, although the support therapy of biological human liver and other organ failure has made progress, but still lack. Specific medical treatment means. In recent years, many advances have been made in the research of the pathogenesis of ACLF, imbalance of immune function, bacterial translocation caused by intestinal bacterial dysplasia and microcirculation disturbance are considered to be an important factor affecting the progress of ACLF, [4]. in liver cirrhosis and severe liver inflammation, the intestinal blood supply and immune state are obviously affected. The changes in the intestinal microenvironment may lead to the imbalance of the intestinal microecological balance. Spontaneous bacterial peritonitis is a common complication of patients with severe hepatitis / chronic acute liver failure. It is one of the common causes of the deterioration of liver function. Intestinal microecological imbalance and bacterial translocation are considered to be the main mechanism leading to the occurrence of SBP, [5].. Over the years, more and more studies have revealed the changes in intestinal flora in chronic liver diseases through 16S rDNA and macrogenome technology, and the intestinal microecological imbalance participates in the development of chronic liver disease and its various complications. However, the intestinal flora of chronic hepatitis B (chronic hepatitis B, CHB) diseases at different stages The changes in the intestinal microflora of hepatitis B related chronic acute liver failure (HBV-ACLF) and CHB disease are still unclear. In order to analyze the intestinal microecological imbalance in CHB patients, explore the characteristics of the structural changes of the intestinal microflora in the HBV-ACLF, in order to understand the pathological mechanism of the intestinal flora in the severe hepatitis. The first part of HBV-ACLF patients' fecal flora diversity study, HBV-ACLF as the research object, chronic hepatitis B severe (severe chronic hepatitis B, S-CHB), chronic hepatitis B mild to moderate (mild and moderate chronic hepatitis) and healthy population as the control group, extraction of feces The sample genome DNA and the 16S rDNA based V3-V4 region high throughput sequencing method were used to analyze the intestinal microflora structure, abundance and diversity of the subjects. The differences and changes of intestinal microflora in the HBV-ACLF and CHB stages were studied. The second part analyzed the ascites culture of patients with spontaneous bacterial peritonitis (SBP) by retrospective analysis. In order to explore the relationship between SBP infection and intestinal flora, and to evaluate the changes of calcitonin (PCT) and blood routine neutrophils in SBP, the main results of this study were to explore the possibility and types of bacteria in negative ascites samples by 16S rDNA high throughput sequencing. The main results were as follows: 1. 16S rDNA high-throughput sequencing of intestinal microflora in patients with severe hepatitis B /HBV-ACLF showed that the number of intestinal flora OTU in HBV-ACLF patients, community richness index were significantly lower than those of M-CHB patients and healthy groups, suggesting that the intestinal flora abundance of HBV-ACLF patients decreased significantly; NMDS analysis showed that HBV-ACLF group could be respectively with M-CHB group, The healthy control group was clearly separated, suggesting that the overall structure of the intestinal flora in HBV-ACLF patients was significantly different from that of the M-CHB and the healthy groups. After the patients with liver cirrhosis were removed, the NMDS analysis could be clearly distinguished, suggesting that the intestinal microflora structure of the patients with HBV-ACLF was significantly changed, and the intestinal flora of the patients with 2.HBV-ACLF was widespread. The community changes, using M-CHB and HBV-ACLF sequenced all bacteria, can distinguish between M-CHB and ACLF patients by random forest analysis, and obtain the rank of the level of the importance of variables. 22 genera, such as 3.Veillonella, Streptococcus, Fusobacterium, were significantly higher in HBV-ACLF patients, and their abundance was significantly positively correlated with TB, INR and MELD scores; Romboutsia and other 26 genera were significantly enriched in M-CHB patients, and their abundance was significantly negatively correlated with TB, INR, and MELD scores, suggesting 48 characteristics. The genera of bacteria are related to the clinical indicators for evaluating the severity of the disease; 4. the succession process of the abundances of the 4. intestinal microflora in the phenotypic state of the acute attack of the chronic hepatitis B shows that the cumulative abundance of the M-CHB- enriched bacteria (MEG) is the potential beneficial bacteria, the cumulative abundance of the ACLF- rich bacteria genus (AEG) is the potential pathogenic bacteria, in the HC group. (MEG was significantly higher than AEG, P0.05), group M-CHB (MEG was significantly higher than AEG, P0.05), S-CHB group (MEG near AEG, P0.05), ACLF group was significantly lower, cumulative abundance was significantly increased, and the abundance ratio of two types of bacteria was inverted; the analysis of 5. spontaneous bacterial peritonitis found that The positive rate of ascites culture in the patients is low, the common bacteria in the ascites culture are Escherichia coli, Klebsiella pneumoniae, Streptococcus, Staphylococcus and so on; the patients with peritoneal irritation sign, but the culture negative ascites samples by 16S rDNA high throughput sequencing preliminary exploration and analysis suggest that one or several bacteria or bacteria DNA may exist in the ascites; ascites detection The species of bacteria were in accordance with the types of potential pathogenic bacteria in the intestinal flora of HBV-ACLF patients. 6. the diagnostic efficacy of the infection index of calcitonin proto (PCT) and blood routine neutrophils in SBP patients was evaluated by ROC curve analysis. The results showed that PCT was in group SBP1 (positive PMN > 250 x 106/L) and SBP2 group (positive PM). N250 * 106/L), the best CUT-OFF values in group SBP3 (culture negative PMN > 250 * 106/L) are: 0.795 ng/m L, 0.265 ng/m L, 0.405 ng/m L; the values are in turn: 100%, 90%, 62.30%; the specificity of specificity is 92.70%, 63.40%, 80.50%, respectively: 92.70%, 62.65%, 65%; The value of UC was as follows: 0.878,0.756,0.669. based on the above boundary value, the proportion of calcitonin and neutrophils in series diagnosis of SBP1, SBP2, SBP3 group AUC value in sequence: 0.976,0.865,0.706. conclusion: This study is based on high throughput sequencing method for the first time to analyze and reveal the characteristics of intestinal microflora disorder in the progressive stages of HBV-ACLF and CHB, with the characteristics of intestinal microflora. CHB disease severity increased, intestinal microflora showed progressive, structural disorder, and overall community abundance decreased significantly; intestinal microflora in HBV-ACLF patients was significantly different from M-CHB patients, the balance of intestinal flora was broken, potential pathogenic bacteria and the proportion of beneficial bacteria abundances were inverted, and the potential pathogenic bacteria in the intestinal flora of HBV-ACLF patients were significantly higher. The species is in accordance with the types of bacteria found in the ascites of SBP patients; this study suggests that the potentially increased potential pathogenic bacteria may increase the risk of infection and play an important role in the development of ACLF, and provide data support for the diagnosis and treatment of ACLF by targeting the intestinal microflora as a target.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R575.3;R512.62

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