基于高通量测序的HBV相关慢加急性肝衰竭患者肠道菌群特征研究

发布时间：2018-04-26 20:08

本文选题：肝炎 + 乙型　；参考：《第三军医大学》2017年硕士论文

【摘要】：慢性乙型肝炎病毒(HBV)感染是一个威胁全球健康的重要公共卫生问题,我国慢性HBV感染者高达约9300万,约有10%-30%的患者出现自发肝炎急性加重(acute exacerbation,AE),部分进展为轻度肝炎,部分进展为重度肝炎以及慢加急性肝衰竭(acute-on-chronic liver failure,ACLF)[1-2]。慢性HBV感染者一旦进展为重型肝炎/ACLF,常合并消化道出血、自发性细菌性腹膜炎(spontaneous bacterial peritonitis,SBP)、肝性脑病等并发症,死亡率极高[3],尽管生物人工肝等器官衰竭的支持治疗已取得进步,但仍缺乏特异的内科治疗手段。最近几年,ACLF的发病机理研究取得诸多进展,免疫功能的紊乱失衡、小肠细菌异常增生所致细菌易位以及微循环障碍被认为是影响ACLF进展的重要因素[4]。在肝硬化及重型肝炎时,肠道血供及免疫状态受到明显影响,肠道微环境改变,可能导致肠道微生态平衡状态发生失衡。自发性细菌性腹膜炎是重型肝炎/慢加急肝衰竭患者的一种常见并发症,是导致肝功能恶化的常见诱因之一,肠道微生态失衡及细菌易位被认为是导致SBP发生的主要机制[5]。近年来,越来越多的研究也通过16S rDNA和宏基因组等技术揭示出肠道菌群在慢性肝脏疾病中的改变,以及肠道微生态失衡参与慢性肝脏疾病及其各种并发症的发生发展。然而关于慢性乙型肝炎(chronic hepatitis B,CHB)疾病不同阶段肠道菌群的变化仍不清,特别是关于乙肝相关慢加急性肝衰竭(HBV-ACLF)和CHB疾病不同严重程度的肠道菌群变化仍然不清楚。为了全面分析CHB患者肠道微生态失衡情况,探索HBV-ACLF的肠道菌群结构变化特征,为理解肠道菌群在重型肝炎病理机制中的作用提供依据。第一部分HBV-ACLF患者粪便菌群结构多样性研究,以HBV-ACLF为研究对象,慢性乙型肝炎重度(severe chronic hepatitis B,S-CHB)、慢性乙型肝炎轻中度(mild and moderate chronic hepatitis B,M-CHB)及健康人群(healthy controls,HC)为对照组,提取粪便样本基因组DNA,运用基于16S rDNA的V3-V4区高通量测序方法分析研究对象的肠道菌群结构、丰度及多样性特点,研究HBV-ACLF和CHB不同疾病阶段肠道菌群的差异及变化特征。第二部分通过回顾性分析自发性细菌性腹膜炎(SBP)患者腹水培养阳性的微生物分布特点、通过16S rDNA高通量测序探索分析培养阴性腹水样本细菌存在的可能性及种类,以期探索SBP感染与肠道菌群的关系,并评估感染标志物降钙素原(PCT)和血常规中性粒细胞在SBP中的变化及诊断效能。本研究主要结果如下:主要结果:1.重型乙型肝炎/HBV-ACLF患者肠道菌群16S rDNA高通量测序结果显示:HBV-ACLF患者肠道菌群OTU数目、community richness指数显著低于M-CHB患者和健康组,提示HBV-ACLF患者肠道菌群丰度显著降低;NMDS分析显示HBV-ACLF组可分别与M-CHB组、健康对照组清晰分开,提示HBV-ACLF患者肠道菌群整体结构显著不同于M-CHB和健康组,去除伴有肝硬化的患者后,NMDS分析仍可清晰区分,提示HBV-ACLF患者无论是否伴有肝硬化其肠道菌群结构均发生显著变化;2.HBV-ACLF患者肠道菌群发生广泛的群落改变,采用M-CHB和HBV-ACLF患者测序得到的所有菌属,通过随机森林分析可显著区分M-CHB和ACLF患者人群,且获得属水平的变量重要性排序。提示采用肠道菌群分类注释的属水平,可显著区分两种疾病状态的患者,不同菌属在区分两组患者时其变量重要性不同;3.Veillonella,Streptococcus,Fusobacterium等22个属在HBV-ACLF患者中显著增高,其丰度升高的程度与TB、INR、MELD评分呈显著正相关;Romboutsia等26个属显著富集在M-CHB患者中,其丰度与TB、INR、MELD评分呈显著负相关,提示48个特征菌属与评价疾病严重程度的临床指标具有相关性;4.肠道菌群特征菌属的丰度在慢乙肝急性发作不同严重程度表型状态下的演替过程显示:M-CHB-富集菌属(MEG)累积丰度作为潜在有益菌,ACLF-富集菌属(AEG)累积丰度作为潜在致病菌,在HC组(MEG显著高于AEG,P0.05),M-CHB组(MEG显著高于AEG,P0.05),S-CHB组(MEG接近AEG,P0.05),ACLF组(MEG显著低于AEG,P0.05);随着疾病的加重MEG累积丰度显著降低,AEG累积丰度显著增高,ACLF患者中两类菌属丰度比例倒置;5.自发性细菌性腹膜炎特点分析发现,SBP患者的腹水培养阳性率低,腹水培养常见细菌为大肠埃希氏菌、肺炎克雷伯菌、链球菌属、葡萄球菌属等;有腹膜刺激征的患者但培养阴性的腹水样本经16S rDNA高通量测序初步探索分析提示可能存在一种或几种细菌或细菌DNA;腹水中检出的细菌种类与HBV-ACLF患者肠道菌群中异常增高的潜在致病菌种类部分一致;6.通过ROC曲线分析评估感染指标血降钙素原(PCT)和血常规中性粒细胞比例在SBP患者中的诊断效能,结果显示PCT在SBP1组(培养阳性PMN≥250×106/L),SBP2组(培养阳性PMN250×106/L),SBP3组(培养阴性PMN≥250×106/L)的最佳CUT-OFF值分别为:0.795 ng/m L、0.265 ng/m L、0.405 ng/m L;AUC值依次为:0.963、0.767、0.714;敏感度依次为:100.00%、90.00%、62.30%;特异度依次为:92.70%、63.40%、80.50%;血常规Neu%诊断的最佳界值分别为:68.45%、62.65%、65.00%;AUC值依次为:0.878、0.756、0.669。依据上述界值,降钙素原和中性粒细胞比例两者串联诊断SBP1、SBP2、SBP3组的AUC值依次为:0.976、0.865、0.706。结论:本研究基于高通量测序方法首次分析并揭示了HBV-ACLF及CHB不同疾病进展阶段的肠道菌群紊乱特征,随着CHB疾病严重程度的增加,肠道菌群呈现进行性、结构性紊乱,整体群落丰度显著降低;HBV-ACLF患者肠道菌群显著区别于M-CHB患者,其肠道菌群的平衡被打破,潜在致病菌和有益菌丰度比例倒置;HBV-ACLF患者肠道菌群中异常增高的潜在致病菌种类与SBP患者腹水中检出的细菌种类部分一致;本研究提示异常增高的潜在致病菌可能增加感染风险从而在ACLF发生、发展过程中扮演重要作用,为通过以肠道菌群为靶点进行ACLF的诊断评估和治疗提供数据支持。
[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.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575.3;R512.62

【参考文献】