【作者】 赵文利；

【导师】 王宇明；

【作者基本信息】 第三军医大学 ， 内科学， 2008， 博士

【摘要】 研究背景:我国肝病临床资源丰富。与西方国家以脂肪肝、酒精性肝炎、丙型肝炎、药物性肝炎和自身免疫性肝病为主的慢性肝病谱不同,慢性乙型肝炎病毒(hepatitis B virus, HBV)感染相关慢性肝病(慢性乙型肝炎、肝硬化、原发性肝癌)一直是我国主要的肝病类型。同时,丙型、甲型、戊型肝炎病毒感染也不容忽视,病毒性肝炎成为我国的优势疾病资源。同时,随着诊断方法和重视程度的提高,自身免疫性肝病的就诊率和诊断率不断上升。自身免疫疾病存在显著的种族差异(发病率、临床表现),如中国人群自身免疫性肝病合并其他系统自身免疫疾病者少见,我国自身免疫性肝病的临床特点及遗传易感性尚未得到充分的研究。鉴定病毒性肝炎和自身免疫性肝炎的遗传因素是一个重要同时又具有挑战性的问题,目前的认识并不充分。对于病毒性肝炎和自身免疫性肝炎这类常见复杂疾病,基于病例-对照设计的遗传关联研究的广泛使用,通常根据生物学功能选择候选基因进行遗传变异与疾病关联的检测。T辅助细胞(Th)极化在病毒性肝炎和自身免疫性肝炎的病理生理过程中发挥关键的作用。近年的研究表明T-bet(由TBX21基因编码)和TIM-1分子是Th细胞极化的关键分子,在病毒感染及自身免疫疾病发病过程中发挥着极其重要的作用。T-bet属于T-box转录因子家族,是调节Th细胞分化的T细胞特异性转录因子,也是CD8+初始T细胞分化为CTL的关键调节因子。T细胞T-bet蛋白水平的下降与机体对一些病毒感染的反应性降低有关,例如HIV感染以及土拨鼠肝炎病毒(woodchuck hepatitis virus, WHV)感染;而T细胞T-bet蛋白水平的升高与炎症反应的结局相关,例如Th1介导的肠炎及肝损伤。人类T-bet蛋白由TBX21基因编码,其常见单核苷酸多态性(single nucleotide polymorphism, SNP)位点位于启动子区。T-1993C位点变异影响TBX21基因的转录活性。T细胞表面免疫球蛋白-黏蛋白样结构蛋白1(T cell immunoglobin domain and mucin domain protein 1,TIM-1)是一种膜表面糖蛋白,表达于CD4+T辅助细胞表面,影响CD4+T辅助细胞发生Th1/Th2极化的关键时期。其在活化的Th2细胞表面高表达,作为共刺激分子,诱导Th2活化增殖。与此同时,TIM-1也是目前已知的甲型肝炎病毒进入细胞的唯一受体。人群中TIM-1基因存在多态性,其外显子4编码的粘蛋白结构域存在插入缺失变异。目的:TBX21和TIM-1基因遗传变异与病毒性肝炎和自身免疫性肝炎的关系值得探讨。本研究基于医院和社区收集病例对照,采用生物学功能候选基因-遗传关联策略,进行Th细胞极化关键基因TBX21和TIM-1与乙型肝炎病毒(hepatitis B virus, HBV)感染、甲型肝炎病毒(hepatitis A virus, HAV)感染和自身免疫性肝炎的遗传关联研究。方法:主要研究工作有:1.在本单位以前的样本库基础上,新收集到1758份人群样本,其中包括男性1241人,女性517人,涵盖了正常对照人群、慢性HBV感染者/自限性清除者、甲型肝炎显性感染患者、隐性感染者、HAV标志物全阴性者以及自身免疫性肝炎患者等临床分组,成功提取DNA。完成新收集到的1758份样本相关病史资料查阅和资料的录入,并对我所前期工作中收集到的4700余例病人病史资料进行完善。对上述病史资料进行整理,剔除重复,获得包括完整病史资料、血DNA和血清的样本4181人份,完成了DNA提取及Panel制作。2.对dbSNP数据库及文献报道的TBX21基因的8个SNP位点,T-1993C、T-1514C、G-1499A、C99G、T1298C、G6547A、T7725C、T9903C,在我们的代表性样本中进行了测序验证及分型方法探索。3.对TIM-1基因外显子4多态性进行了克隆-测序发掘,构建了单倍型,并摸索了157MVTTTP插入缺失多态性的GeneScan分型方法。4.选择了TBX21基因T-1993C和TIM-1基因157MVTTTP插入缺失多态位点,对正常对照人群、慢性HBV感染者/自限性清除者、甲型肝炎显性感染患者、隐性感染者、HAV标志物全阴性者以及自身免疫性肝炎患者进行了基因分型及疾病关联分析。结果:主要研究结果有:1.重庆人群中,TBX21基因外显子区和启动子区的最高频多态位点是T-1993C,国外报道的G-1499A和T9903C位点频率极低(等位频率< 1%)。2.重庆人群中,TIM-1基因外显子4有高度多态性,主要单倍型有4种。3.以无症状携带者为对照,TBX21基因T-1993C位点与慢性HBV感染疾病进程无显著关联。以隐性感染者为对照,TBX21基因T-1993C位点与HAV感染疾病状态无显著关联。4.以慢性HBV感染者为对照,TBX21基因T-1993C位点与HBV自限性清除存在显著的关联(χ2 = 20.4, P = 0.0000063);启动子区单倍型-1993T-1514T显著降低慢性HBV感染的风险(P = 0.000017, OR = 0.46, 95% CI 0.32～0.65)。5.以献血员为对照,TBX21基因T-1993C位点与自身免疫性肝炎存在显著关联,-1993T等位与自身免疫性肝炎显著相关(χ2 = 13.7, P = 0.00004)。多因素Logistic回归校正年龄、性别等因素,-1993T/T纯合子发生自身免疫性肝炎的风险度显著升高(P=0.00043,OR=0.26,95%CI=0.12～0.55)。6. TIM-1基因157MVTTTP插入缺失多态位点对HBV感染、HAV感染和自身免疫性肝炎的遗传风险度没有显著影响。结论:我们基于医院的病例对照研究结果表明,TBX21基因T-1993C位点与HBV自限性清除及自身免疫性肝炎相关联。根据文献报道,疾病关联的T-1993C位点是TBX21基因启动子的潜在调节性SNP位点,其遗传变异影响TBX21基因的转录活性,-1993C等位的转录活性较强。有关TBX21基因T-1993C位点与HBV自限性清除及自身免疫性肝炎的遗传关联及机制,值得进一步深入研究。更多还原

【Abstract】 Background: Liver diseases are common in China. The major types of liver diseases are hepatitis B virus (HBV) related disorders (such as chronic hepatitis B, liver cirrhosis and hepatocellular carcinoma), it is different from liver disease spectrum in Caucasians, in which lipid/alcoholic hepatitis, drug induced hepatitis, hepatitis C and autoimmune liver diseases are more common. Beside this, hepatitis A/C/E virus infection also need to be attention. Followed with the improvement of diagnostics and emphasis level, the autoimmune liver diseases are highly recognized and treated in recent years. The prevalence and some clinical characteristics of autoimmune diseases are significantly different among different ethnic populations, e.g, combination autoimmune manifestion with other organs are rare in Chinese patients. However, the clinical characteristics and host genetic factors of autoimmune hepatitis have not been fully analysed.It is an important and challenging issue for identification of host genetic factors involved in viral hepatitis and autoimmune hepatitis, which has not been fully understand. For such common complex diseases, genetic association studies are widely used, based on case control design. Genetic associations are detected between biological functional candidate genes and diseases. T helper (Th) cells polarization play a key role in the pathogenetic process of viral infection and autoimmune hepatitis.T-bet is a member of the T-box family of transcription factors, which is responsible for the induction of Th1 cells and the repression of Th2 cells from naive T lymphocytes. Reduced T-bet concentrations have been linked with diminished responses to some viral infections, including human immunodeficiency virus (HIV) infection and woodchuck hepatitis virus (WHV) infection, and higher levels have been linked with inflammatory outcomes such as Th1-mediated colitis and liver injury in animal models. The human TBX21 gene encoding T-bet, some common single nucleotide polymorphisms (SNPs) are located in the promoter region. T-1993C is a functional site which may influence the transcriptional activity of TBX21 promoter.T-cell immunoglobulin domain and mucin domain protein 1 (TIM-1) is a glycoprotein, which is expressed on the surface of CD4+ Th cells. It plays an important role on the Th1/Th2 polarization. TIM-1 is highly expressed on the surface of Th2 cells as a co-stimulation molecule and induces the activation and proliferation of Th2 cells. TIM-1 is also the only cellular receptor for hepatitis A virus (HAV). TIM-1 is polymorphic in population, there is a 18-bp insertion variation in exon 4 (mucin domain).Objective: Because the expression of TBX21 and TIM-1 are important for Th1/Th2 immune response, it is interesting to examine whether polymorphisms of TBX21 and TIM-1 genes are associated with HAV/HBV infection and autoimmune hepatitis. In this study, we collected cases and controls samples based on hospital or community, then conducted genetic association studies between TBX21/TIM-1 genes and HAV infection, HBV infection and autoimmune hepatitis.Methods: We completed the following works:1. We newly collected 1758 cases and controls (1241 males and 517 females), including normal healthy controls, patients with chronic HBV infection or spontaneously clearance, patients with hepatitis A infection, cases with asymptomatic HAV infection, cases negative for HAV markers and patients with autoimmune hepatitis. Clinical and epidemiological information were collected, genomic DNA was extracted. Along with previous samples, we made DNA panels.2. Eight known SNPs of TBX21, the T-1993C, T-1514C, G-1499A, C99G, T1298C, G6547A, T7725C and T9903C, were validated in our representative samples, appropriate genotyping methods were developed.3. The polymorphisms in exon 4 of TIM-1 were discovered by TA-clone and sequencing, haplotypes were constructed, and the GeneScan genotyping method for 157MVTTTP insertion/deletion was established.4. TBX21 T-1993C and TIM-1 157MVTTTP insertion/deletion polymorphisms were selected for genotyping in normal healthy controls, patients with chronic HBV infection or spontaneously clearance, patients with hepatitis A infection, cases with asymptomatic HAV infection, cases negative for HAV markers and patients with autoimmune hepatitis. Genetic association analysis were conducted.Results: Based on studies mentioned above, we demonstrated the following main results:1. T-1993C is the most common polymorphism in the exons and promoter region of TBX21. Previously reported G-1499A and T9903C SNP had very low frequencies (< 1%) in Chongqing population.2. The exon 4 of TIM-1 is highly polymorphic in Chongqing population. There were four major haplotypes in Chongqing population.3. Compared with asymptomatic HBV carriers, TBX21 T-1993C was not associated with disease progression of chronic HBV infection. Compared with cases with asymptomatic HAV infection, TBX21 T-1993C was not associated with HAV infection.4. Compared with patients of chronic HBV infection, TBX21 T-1993C was significantly associated with spontaneously clearance of HBV (χ2 = 20.4, P = 0.0000063). Similarly, haplotype -1993T-1514T was associated with decreased susceptibility to the persistence of HBV infection (P = 0.000017, OR = 0.46, 95% CI 0.32-0.65).5. Compared with blood donors, TBX21 -1993T allele was significantly associated with autoimmune hepatitis (χ2 = 13.7, P = 0.00004). By logistic regression controlling confounding factors such as age and sex, -1993T/T homozygotes had a significantly increased risk for autoimmune hepatitis (P=0.00043, OR=0.26, 95%CI = 0.12-0.55).6. TIM-1 157MVTTTP insertion/deletion polymorphism had no significant influence on the genetic risks for HAV infection, HBV infection or autoimmune hepatitis.Conclusion: In conclusion, based on case control collection, polymorphic loci screening and genotyping method establishment, we selected TBX21 T-1993C and TIM-1 157MVTTTP insertion/deletion polymorphisms for genotyping in our large case control samples. We detected the genetic associations between TBX21/TIM-1 genes and HAV infection, HBV infection and autoimmune hepatitis. Our case control association study indicated that TBX21 T-1993C was associated with HBV clearance and autoimmune hepatitis. Previous luciferase reporter assays and electrophoretic mobility shift assays suggest T-1993C polymorphism affects the transcriptional activity of TBX21 and the -1993C allele may contribute to an increase in T-bet expression. Nevertheless, more steps are required before the importance of the TBX21 gene in HBV infection and autoimmune hepatitis can be fully ascertained. First, data from other ethnic populations are needed to confirm our initial observation. Second, the promoter polymorphisms in the TBX21 gene and its functional molecular mechanisms for this association with diseases should be identified.更多还原