本文選題：慢加急性肝衰竭 + 人類白細(xì)胞抗原�。� 參考：《第三軍醫(yī)大學(xué)》2016年博士論文

【摘要】：HBV相關(guān)慢加急性肝衰竭(HBV-related acute-on-chronic liver failure,HBV-ACLF)是我國及亞太地區(qū)慢性肝病患者常見的危急重癥,臨床上表現(xiàn)為短期內(nèi)出現(xiàn)高膽紅素血癥、凝血酶原時(shí)間延長(INR≥1.5),起病迅速,病死率高,缺乏特異治療手段。目前認(rèn)為HBV-ACLF的發(fā)生主要與宿主遺傳因素、病毒以及宿主免疫相互作用有關(guān),但是其確切的發(fā)病機(jī)制尚不明確。近年來,西方基于酒精性肝硬化的ACLF的研究報(bào)道較多,認(rèn)為酒精性肝硬化相關(guān)ACLF患者從急性發(fā)作、炎癥損傷、免疫失調(diào)到系統(tǒng)性炎癥反應(yīng)綜合征(systematic inflammatory response syndrome,SIRS)、代償性抗炎反應(yīng)綜合征(compensatory anti-inflammatory response syndrome,CARS)、再生反應(yīng)等多環(huán)節(jié)事件的幅度和次序,決定著患者是炎癥消退-康復(fù)還是進(jìn)展到肝外器官衰竭,系統(tǒng)性炎癥反應(yīng)是酒精性肝硬化患者發(fā)生ACLF的主要驅(qū)動因素。慢性HBV感染者是否發(fā)生肝炎發(fā)作,發(fā)作后是否進(jìn)展為肝衰竭,由于HBV缺乏慢性感染的動物模型,且ACLF的病因和誘因存在很大的異質(zhì)性,目前對HBV相關(guān)ACLF的肝臟免疫學(xué)應(yīng)答及免疫病理過程仍然知之甚少。既往針對HBV病毒序列變異的研究認(rèn)為C基因啟動子區(qū)、前C區(qū)和C區(qū)變異與乙型肝炎的發(fā)作及暴發(fā)性肝衰竭相關(guān)。但是,同樣的前C、C區(qū)變異既可以見于不同的病變表型(普通乙型肝炎、肝硬化、肝癌),也可見于無癥狀攜帶者。事實(shí)上,病毒變異是否導(dǎo)致乙型肝炎重癥化的發(fā)生和發(fā)展,受到宿主因素的制約。乙型肝炎的發(fā)生、發(fā)展和轉(zhuǎn)歸,是病毒(病毒載量、變異、進(jìn)化)和宿主(遺傳異質(zhì)性、年齡、性別等)通過免疫應(yīng)答相互作用導(dǎo)致的常見復(fù)雜疾病。常見復(fù)雜疾病(common complex diseases)通常由許多微效累加基因與環(huán)境因素共同作用而決定的。HBV-ACLF的發(fā)生、發(fā)展,涉及到免疫識別、炎癥活化/放大、肝細(xì)胞壞死、SIRS、CARS、器官衰竭等多個(gè)環(huán)節(jié),也屬于常見復(fù)雜疾病的范疇,其遺傳因素也涉及多個(gè)微效基因的作用。在復(fù)雜疾病遺傳易感性研究中,關(guān)聯(lián)研究-連鎖不平衡分析方法最為常用,統(tǒng)計(jì)效能也遠(yuǎn)高于家系連鎖分析。基于全基因組策略的遺傳關(guān)聯(lián)研究,是目前復(fù)雜疾病遺傳關(guān)聯(lián)研究的主流,與候選基因策略相比其優(yōu)點(diǎn)在于統(tǒng)計(jì)效能有著極大的提高,同時(shí)可以避免群體分層偏倚和基因選擇的偶然性,可以從全基因組角度獲得復(fù)雜疾病遺傳特征的全局、系統(tǒng)的認(rèn)識。開展HBV-ACLF表型的全基因組關(guān)聯(lián)研究(genome-wide association study,GWAS),解析其全基因組范圍內(nèi)的宿主遺傳位點(diǎn)及遺傳關(guān)聯(lián)圖譜,有助于認(rèn)識HBV-ACLF的驅(qū)動機(jī)制及干預(yù)靶點(diǎn)。因此,本課題在建立HBV-ACLF病例大樣本、多中心隊(duì)列的基礎(chǔ)上,對其臨床特征及炎癥因子譜進(jìn)行了分析,開展了HBV-ACLF的全基因組關(guān)聯(lián)研究,并對主要關(guān)聯(lián)位點(diǎn)HLA-DR進(jìn)行了精細(xì)解析和功能注釋,以及陽性關(guān)聯(lián)HLA等位對HBV變異的限制性分析。本研究結(jié)果首次從全基因組角度明確了HBV-ACLF的主要遺傳位點(diǎn),提示HLA-DR等位限制性CD4+T細(xì)胞途徑對HBV-ACLF的驅(qū)動作用。本課題的主要研究結(jié)果如下:1.共納入1300例HBV-ACLF患者和2087例HBV無癥狀攜帶者進(jìn)入3-stage的全基因組關(guān)聯(lián)研究,樣本來自重慶、北京、泉州、遵義、南寧5個(gè)中心。所有患者均無抗病毒史、無既往肝炎發(fā)作或肝功能失代償病史。對1300例HBV-ACLF患者進(jìn)行臨床特征分析,LC-ACLF占60%左右,NLC-ACLF占40%;HBe Ag陰性的HBV-ACLF占60%,HBe Ag陰性并且HBV DNA104 IU/m L的HBV-ACLF占17.1%。與LC-ACLF相比,NLC-ACLF患者表現(xiàn)為更年輕(平均年齡39.5 vs.43.9歲,p=3.14×10-11)、更高的ALT水平(平均值1153 vs.809 IU/L,p=1.36×10-11)、腹水陽性率較低(58.7%vs.88.4%,p=9.25×10~(-35))、起病后更短時(shí)間內(nèi)INR達(dá)到1.5及發(fā)生腦病(平均值分別19.8vs.24.8天,p=1.36×10-11;27.3 vs.38.1天,p=5.16×10-6)。2.對1013份患者血清樣本進(jìn)行35種細(xì)胞因子的多重定量檢測,結(jié)果顯示,國外酒精性肝硬化ACLF研究所關(guān)注的重要指標(biāo)PCT(提示細(xì)菌感染)和CRP(提示肝外器官衰竭),在我們HBV-ACLF患者中其濃度低于普通慢性乙型肝炎(CHB)患者。與慢性乙型肝炎輕中度及重度相比,HBV-ACLF患者顯著升高的細(xì)胞因子有6種:Th1型細(xì)胞因子IL-1a、TNF-β,Th2型細(xì)胞因子IL-5、IL-10和IL-13,Th17型細(xì)胞因子IL-17。3.HBV-ACLF的GWAS初始階段分析顯示全基因組范圍內(nèi)1、6、12號染色體有顯著的關(guān)聯(lián)信號(p1×10-5),LC-ACLF和NLC-ACLF兩種亞型的共同遺傳關(guān)聯(lián)位點(diǎn)位于6號染色體HLA-II區(qū)域。經(jīng)過replication 1a和1b獨(dú)立樣本驗(yàn)證表明HLA-DR區(qū)域是主要關(guān)聯(lián)位點(diǎn),再經(jīng)過replication 2a和2b驗(yàn)證,明確了rs3129859與HBV-ACLF顯著相關(guān)(P combine=7.40×10-19,OR=1.83)。4.分層分析顯示rs3129859是HBV-ACLF的獨(dú)立風(fēng)險(xiǎn)因素,與慢性乙型肝炎活動狀態(tài)和HBV再活化無關(guān)。風(fēng)險(xiǎn)等位rs3129859*C與HBV-ACLF臨床進(jìn)程相關(guān),攜帶風(fēng)險(xiǎn)等位rs3129859*C的ACLF患者和S-CHB患者,在入院28天時(shí)INR達(dá)到1.5及發(fā)生腹水的風(fēng)險(xiǎn)更高(分別有p=3.95×10-4,p=3.03×10-4)。在NLC-ACLF亞組,攜帶風(fēng)險(xiǎn)等位rs3129859*C的患者,28天死亡率更高(p=0.03)。5.HLA-DRB1*1202等位是HBV-ACLF最顯著的風(fēng)險(xiǎn)等位(p=3.94×10-6,OR=2.05)。HLA-DRB1*1202的全球分布與HBV-ACLF地理流行病學(xué)趨勢一致,在中國(5.9-21.7%)和東南亞地區(qū)(6.8-35.3%)等位頻率最高。6.HLA-DRβ第28位氨基酸是最顯著的關(guān)聯(lián)氨基酸(p=3.03×10-6,OR=1.75)。其次,關(guān)聯(lián)的氨基酸還包括HLA-DRβ蛋白第26、30、32、37、38和85位氨基酸,以及HLA-DQα蛋白第40、47、50、51、53、56、69、76位氨基酸和HLA-DQβ蛋白第45位氨基酸(p0.001)。蛋白結(jié)構(gòu)三維重建顯示這些關(guān)聯(lián)氨基酸位于HLA分子抗原結(jié)合溝槽內(nèi)。7.等位單倍型rs3129859C-DRB1*1202-DQA1*0601-DQB1*0301和氨基酸單倍型DRβ-LEHHLLA-DQα-GCVLQdel TL-DQβ-E是風(fēng)險(xiǎn)單倍型,攜帶風(fēng)險(xiǎn)單倍型的HBV感染者,罹患ACLF的風(fēng)險(xiǎn)更高(additive model,分別p=1.16×10-4,OR=1.94;p=2.43×10-6,OR=2.10)。8.生存分析提示風(fēng)險(xiǎn)等位及風(fēng)險(xiǎn)單倍型與HBV-ACLF臨床進(jìn)程相關(guān)。在非肝硬化ACLF亞組,攜帶風(fēng)險(xiǎn)單倍型rs3129859C-DRB1*1202-DQA1*0601-DQB1*0301的患者入院后28天(p=3.49×10-4)、90天(p=0.003)及180天(p=0.003)的死亡率更高,且肝性腦病發(fā)生率更高(p=0.019),氨基酸單倍型也觀察到一致的相關(guān)性。9.HBV-ACLF關(guān)聯(lián)位點(diǎn)功能注釋:e QTL分析顯示關(guān)聯(lián)位點(diǎn)rs3129859與HLA-II基因表達(dá)相關(guān);GRAIL文獻(xiàn)分析顯示HLA-DR和HLA-DQ是最合理的關(guān)聯(lián)基因。與其他HBV相關(guān)GWAS文獻(xiàn)overlap分析,顯示HBV-ACLF的遺傳關(guān)聯(lián)位點(diǎn)與慢性HBV感染的遺傳關(guān)聯(lián)位點(diǎn)有部分重疊。10.通過分析HLA-DRB1*1202風(fēng)險(xiǎn)等位對HBV變異的限制作用,鑒定出12個(gè)HBV變異,既表現(xiàn)出HBV-ACLF風(fēng)險(xiǎn)等位HLA-DRB1*1202特異性和肝炎活化特異性,同時(shí)也是病毒發(fā)生正選擇的熱點(diǎn)氨基酸位置。表位預(yù)測分析發(fā)現(xiàn),這些變異可引發(fā)病毒表位漂移。結(jié)論:1.我們的數(shù)據(jù)對于認(rèn)識HBV-ACLF的自然史有重要意義:相當(dāng)比例的慢性HBV感染者可在無肝硬化基礎(chǔ)的情況下發(fā)生ACLF;HBV自然史處于低復(fù)制期和e抗原陰性期的患者,發(fā)生ACLF的風(fēng)險(xiǎn)不容忽視;NLC-ACLF的疾病進(jìn)展更為迅速,HBV-ACLF發(fā)病年齡(42歲)遠(yuǎn)低于歐洲酒精性肝硬化ACLF患者(56歲)。2.HBV-ACLF患者血清炎癥因子譜分析表明HBV-ACLF患者確實(shí)存在劇烈的SIRS和CARS并存局面。HBV-ACLF患者CD4 Th細(xì)胞因子通路劇烈活化,有別于慢性乙型肝炎輕中度和重度,也不同于歐洲酒精性肝硬化ACLF患者。3.NLC-ACLF和LC-ACLF亞型具有共同的遺傳易感位點(diǎn),亞型位點(diǎn)、定量性狀位點(diǎn)(肝酶、性激素等)與HBV-ACLF位點(diǎn)通路存在交互作用。4.HLA-DR是HBV-ACLF的主要遺傳易感位點(diǎn),風(fēng)險(xiǎn)等位rs3129859*C和HLA-DRB1*1202可作為HBV-ACLF臨床預(yù)警預(yù)后的marker。風(fēng)險(xiǎn)等位有特異限制的病毒變異位點(diǎn),與肝炎活化、病毒正選擇和表位漂移有關(guān)�？傊�,本研究首次在HBV相關(guān)慢加急性肝衰竭患者中開展了全基因組關(guān)聯(lián)研究,從全基因組范圍內(nèi)鑒定出HLA-DR是HBV-ACLF的主要關(guān)聯(lián)位點(diǎn),且關(guān)聯(lián)的風(fēng)險(xiǎn)等位和單倍型與HBV-ACLF臨床進(jìn)程顯著相關(guān),提示了HLA-II類分子限制的CD4+T細(xì)胞途徑在HBV-ACLF的免疫病理過程中的重要作用,同時(shí)也表明肝硬化和非肝硬化基礎(chǔ)的HBV-ACLF為同質(zhì)性疾病,支持一個(gè)疾病的假說(one disease hypothesis)。
[Abstract]:HBV related chronic acute liver failure (HBV-related acute-on-chronic liver failure, HBV-ACLF) is a common critical disease in the patients with chronic liver disease in China and the Asia Pacific region. The clinical manifestations include hyperbilirubinemia in the short term, prolonged prothrombin time (INR > 1.5), rapid onset, high mortality, and lack of specific treatment. At present H The occurrence of BV-ACLF is mainly related to the host genetic factor, virus and host immune interaction, but its exact pathogenesis is not clear. In recent years, there are many reports on ACLF based on alcoholic cirrhosis in the West. It is considered that alcoholic cirrhosis related ACLF patients are from acute attack, inflammatory injury, immune disorder to systemic inflammation. The response syndrome (systematic inflammatory response syndrome, SIRS), compensatory anti inflammatory response syndrome (compensatory anti-inflammatory response syndrome, CARS), regenerative response, and the magnitude and order of the multiple link events determine that the patient is retreated by inflammation or progressing to extrahepatic organ failure, and the systemic inflammatory response is wine The main driving factors of ACLF in patients with severe cirrhosis. Whether or not chronic HBV infected persons have HBV attack, whether or not they are progressing to liver failure after the seizure, because HBV lacks the animal model of chronic infection, and the causes and causes of ACLF are very heterogeneous. The liver immunological response and immunopathological process of HBV related ACLF are still known to be still known. Very few. Previous studies of HBV virus sequence variation suggest that the C gene promoter region, the anterior C region and the C region variation are associated with the attack of hepatitis B and fulminant liver failure. However, the same pre C, C region variation can be seen in different pathological phenotypes (common hepatitis B, liver hardened, liver cancer), or asymptomatic carriers. In fact, the disease is a disease. Whether the virus variation leads to the occurrence and development of hepatitis B severe and is restricted by host factors. The occurrence, development and prognosis of hepatitis B are common complex diseases caused by the interaction of virus (viral load, variation, evolution) and host (genetic heterogeneity, age, sex, etc.) through immune response. Common complex diseases (common complex) Diseases) the occurrence and development of.HBV-ACLF, which is usually determined by the interaction of many micro effect accumulating genes and environmental factors, involves immune recognition, inflammatory activation / amplification, hepatocyte necrosis, SIRS, CARS, organ failure and so on. It also belongs to the common complex disease domain. Its genetic factors also involve the role of multiple micro genes. In the study of genetic susceptibility to miscellaneous diseases, association studies - linkage disequilibrium analysis is the most common method, and the statistical efficiency is far higher than that of family linkage analysis. Genetic association based on the whole genome strategy is the mainstream of the genetic association of complex diseases, and the advantages of the genetic association are that the statistical efficiency is greatly improved. At the same time, it can avoid the chance of population stratification bias and gene selection. It can obtain the global and systematic understanding of the genetic characteristics of complex diseases from the whole genome angle. The whole genome association study of the HBV-ACLF phenotype (genome-wide association study, GWAS) is used to analyze the host genetic locus and genetic correlation within the whole genome. The combined atlas helps to understand the driving mechanism of HBV-ACLF and the target of intervention. Therefore, on the basis of the establishment of a large sample of HBV-ACLF cases and multi center queues, the clinical features and the spectrum of inflammatory factors are analyzed, and the whole genome association of HBV-ACLF is carried out, and the main associated locus HLA-DR is carefully parsed and functional. Annotations, and the restrictive analysis of HBV variation with positive associated HLA alleles. The results of this study have first identified the main genetic locus of HBV-ACLF from the whole genome, suggesting the driving effect of the HLA-DR allelic restrictive CD4+T cell pathway on HBV-ACLF. The main results of this study are as follows: 1. a total of 1300 patients with HBV-ACLF and 2087 cases of HBV were included. A complete genome association study of asymptomatic carriers entered 3-stage, with samples from 5 centers in Chongqing, Beijing, Quanzhou, Zunyi and Nanning. All patients had no history of antiviral, no previous hepatitis and liver function decompensation. 1300 cases of HBV-ACLF patients were analyzed, LC-ACLF accounted for 60%, NLC-ACLF accounted for 40%, and HBe Ag negative HBV- ACLF was 60%, HBe Ag was negative and HBV-ACLF of HBV DNA104 IU/m L accounted for 17.1%. compared to LC-ACLF. NLC-ACLF patients showed younger (average age 39.5 vs.43.9, 10-11), higher level (average value 1153, 10-11). R reached 1.5 and the occurrence of encephalopathy (average 19.8vs.24.8 days, p=1.36 x 10-11; 27.3 vs.38.1 days, p=5.16 * 10-6).2. multiple quantitative detection of cytokines in 1013 serum samples. The results showed that the important index of foreign alcoholic liver cirrhosis ACLF research, PCT (suggestive of bacterial infection) and CRP (suggestive of liver organ failure) In our HBV-ACLF patients, their concentration was lower than that of patients with chronic hepatitis B (CHB). Compared with mild to moderate and severe chronic hepatitis B, there were 6 significant cytokines in HBV-ACLF patients: Th1 cytokine IL-1a, TNF- beta, Th2 cytokine IL-5, IL-10 and IL-13, Th17 cytokine IL-17.3.HBV-ACLF. The analysis showed that there was a significant correlation signal (P1 x 10-5) on chromosome 1,6,12 in the whole genome, and the common genetic association loci of the two subtypes of LC-ACLF and NLC-ACLF were located in the HLA-II region of chromosome 6. The independent sample of replication 1a and 1b showed that the HLA-DR region was the main Association site, and then confirmed by replication 2a and 2b. The significant correlation between rs3129859 and HBV-ACLF (P combine=7.40 x 10-19, OR=1.83).4. stratification analysis showed that rs3129859 was an independent risk factor for HBV-ACLF, not related to chronic hepatitis B activity state and HBV reactivation. Risk allele rs3129859*C was associated with HBV-ACLF clinical processes, carrying patients with risk allele and patients, At 28 days, the risk of INR reached 1.5 and the risk of ascites was higher (p=3.95 x 10-4, p=3.03 x 10-4). In the NLC-ACLF subgroup, the 28 day mortality (p=0.03).5.HLA-DRB1*1202 was the most significant risk allele of HBV-ACLF (p= 3.94 x 10-6, OR=2.05).HLA-DRB1*1202 (p= 3.94 x 10-6, OR=2.05).HLA-DRB1*1202. The trend of geographical epidemiology is consistent. The highest.6.HLA-DR beta twenty-eighth - bit amino acids in China (5.9-21.7%) and Southeast Asia (6.8-35.3%) are the most significant associated amino acids (p=3.03 x 10-6, OR=1.75). Secondly, the associated amino acids also include HLA-DR beta protein 26,30,32,37,38 and 85 - bit amino acids, as well as HLA-DQ alpha protein 40,47,50,51,53 56,69,76 amino acid and HLA-DQ beta protein forty-fifth amino acid (p0.001). The three-dimensional reconstruction of protein structure shows that these associated amino acids are located at the.7. allele of the HLA molecular antigen binding groove, the.7. allele rs3129859C-DRB1*1202-DQA1*0601-DQB1*0301 and the amino acid haplotype DR beta -LEHHLLA-DQ a -GCVLQdel TL-DQ beta -E are the risk haplotypes, carrying the risk. The risk of ACLF was higher in haplotype HBV infected people (additive model, p=1.16 x 10-4, OR=1.94; p=2.43 x 10-6, OR=2.10).8. survival analysis suggested that the risk allele and the risk haplotype were related to the HBV-ACLF clinical process. In the non cirrhosis ACLF subgroup, the patients with the risk of the risk of the double type rs3129859C-DRB1*1202-DQA1*0601-DQB1*0301 were hospitalized. The mortality of the 28 days (p=3.49 x 10-4), 90 days (p=0.003) and 180 days (p=0.003) was higher, and the incidence of hepatic encephalopathy was higher (p=0.019). The amino acid haplotype also observed the consistent correlation of.9.HBV-ACLF associated site function annotation: e QTL analysis showed that the associated locus rs3129859 was associated with the HLA-II gene expression; GRAIL literature analysis showed HLA-DR and HLA. -DQ is the most reasonable association gene. Overlap analysis with other HBV related GWAS literature shows that there is partial overlap between the genetic association site of HBV-ACLF and the genetic association loci of chronic HBV infection, and.10. is limited by the analysis of HLA-DRB1*1202 risk alleles, identifying 12 HBV variations, showing both HBV-ACLF risk allele HLA-DRB1*120. 2 specificity and specificity of hepatitis activation are also the location of positive hot amino acids that the virus is selecting. Epitope prediction analysis found that these variations can cause virus epitopes drift. Conclusion: 1. our data are important for the understanding of the natural history of HBV-ACLF: a considerable proportion of chronic HBV infections can be in the absence of liver cirrhosis. ACLF, HBV natural history was at low replication and e antigen negative, the risk of ACLF could not be ignored; NLC-ACLF's disease progressed more rapidly. The age of HBV-ACLF (42 years old) was far below the European alcoholic liver cirrhosis (56 years old) ACLF patients (56 years old), the serum inflammatory factor analysis of.2.HBV-ACLF patients showed that the HBV-ACLF patients did have acute severity. The coexistence of SIRS and CARS in.HBV-ACLF patients with CD4 Th cytokine pathway is strongly activated, different from chronic hepatitis B, moderate and severe, and the.3.NLC-ACLF and LC-ACLF subtypes of ACLF patients in European alcoholic cirrhosis have common genetic susceptibility loci, subtype loci, and quantitative trait loci (liver enzymes, sex hormones, etc.) and HBV-ACLF bits. .4.HLA-DR is the main genetic susceptibility locus of HBV-ACLF, and risk allele rs3129859*C and HLA-DRB1*1202 can be used as the marker. risk allele of HBV-ACLF clinical early warning prognosis, which is related to hepatitis activation, positive virus selection and epitope drift. All genomic association studies have been carried out in patients with acute hepatic failure to identify HLA-DR as the main associated locus of HBV-ACLF, and the associated risk alleles and haplotypes are significantly related to the clinical process of HBV-ACLF, suggesting that the CD4+T cell pathway restricted by the HLA-II class is important in the immune pathological process of HBV-ACLF. It also indicates that HBV-ACLF is a homogenous disease of liver cirrhosis and non cirrhosis. It supports the hypothesis of a disease (one disease hypothesis).
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R512.62;R575.3
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本文編號：1902684