【作者】 葛剑；

【导师】 王凯；

【作者基本信息】 山东大学 ， 内科学， 2010， 博士

【摘要】 第一部分Th17、Th1和Tc1细胞亚群在慢性乙型肝炎患者中的变化及意义慢性乙型肝炎的发病机制十分复杂,宿主免疫调节的紊乱是导致病毒不能有效清除、病情迁延不愈、引起慢性炎症、造成肝脏病变的重要原因。许多研究表明Th1、Th2细胞亚群的比例失衡、功能失调是导致慢性乙肝发病的重要因素。传统上,CD4+辅助T细胞(Th)和CD8+细胞毒性T细胞(Tc)根据各自分泌的细胞因子的不同,可以将T细胞分为两类：Ⅰ类T细胞(Th1和Tc1,合称T1)和Ⅱ类T细胞(Th2和Tc2,合称T2),前者主要分泌干扰素γ(IFN-γ)、白细胞介素(IL)-2、肿瘤坏死因子(TNF)-β,后者主要分泌IL-4、IL-5、IL-6、IL-10和IL-13,并且常以IFN-γ/IL-4代表T1／T2的比例。T1和T2在免疫调节中均发挥重要作用,它们各自分泌的细胞因子对免疫应答效应起关键作用。CD8+T细胞可分为Tc1和Tc2两个细胞亚群,Tc1与Th1亚群相似,可分泌IL-2、IFN-γ等细胞因子并且可发挥细胞毒作用。Th17细胞是最近发现的一种新的效应性CD4+T细胞亚群,以产生分泌白介素17(IL-17)为特征。Th17细胞与Th1和Th2细胞无论在分化途径、信号转导还是生物学功能等方面均有所不同。大量证据表明Th17细胞通过产生IL-17作用于不同的靶细胞,诱导其他细胞因子产生,参与细胞因子网络调节,触发炎症递质释放,从而介导炎性反应(防御胞外病原菌的感染)、自身免疫性疾病、肿瘤和移植排斥等病理状态的发生和发展。迄今为止,有关慢性乙型肝炎患者外周血中Th17细胞的研究报道很少。目的本研究的目的是通过对慢性乙型肝炎患者外周血Th17、Th1和Tc1细胞亚群及血浆细胞因子的检测,进一步探讨三种细胞亚群在慢性乙型肝炎患者发病机制中的作用及评价它们的临床相关性。方法应用三色流式细胞术检测慢性乙型肝炎患者和正常对照外周血中Th1、Th17和Tc1细胞占CD3+T细胞的比例。三种细胞亚群的界定分别为：Th17细胞为CD3+CD8-IL-17A+,Th1细胞为CD3+CD8-IFN-γ+,Tc1细胞为CD3+CD8+IFN-γ+；应用ELISA技术检测慢性乙型肝炎患者和正常对照血浆中IL-17和IFN-γ的表达水平。应用逆转录聚合酶链反应(RT-PCR)方法检测IL-17及IFN-γ在慢性乙型肝炎患者和正常对照外周血单个核细胞中的表达。结果1、慢性乙型肝炎患者外周血Th1细胞的比例(8.55±1.53％)明显低于正常对照组(10.56±1.91％)(P<0.01),并且慢性乙型肝炎患者Tc1细胞的比例(6.35±1.85％)较正常对照组(9.0±2.79％)亦显著降低(P<0.05)。2、慢性乙型肝炎患者Th17细胞的比例(1.53±0.52％)较正常对照(0.92±0.20％)显著升高(P<0.05)。3、在慢性乙型肝炎患者中,Th1与Tc1细胞间存在明显的正相关性(r=0.58,P<0.05),Th17与Thl细胞间亦存在显著的负相关性(r=-0.56,P<0.05),但Th17与Tc1细胞间无相关性。4、慢性乙型肝炎患者血浆中IFN-γ和IL-17表达水平与正常对照相比均无显著性差异。5、在慢性乙型肝炎患者中,外周血中Th17细胞亚群的比例与ALT呈明显正相关(r=0.66,P<0.05)。慢性乙型肝炎患者外周血中Th1细胞亚群的比例与ALT呈显著负相关(r=-0.68,P<0.05)。结论我们的研究结果进一步表明,在慢性乙型肝炎的异常免疫反应中,Th1和Tc1型免疫反应受到抑制。尤为重要的是,我们研究首次表明,在慢性乙型肝炎发生和发展中,Th17细胞亚群比例增加可能是一个重要的决定因素,调节慢性乙型肝炎患者中Th17细胞的异常可能成为慢性乙型肝炎治疗的新策略。第二部分Th17、Th1和Tc1细胞亚群在慢加急性重型乙型病毒性肝炎患者中的变化及意义HBV感染可引起人类急慢性肝炎,与肝硬化和肝细胞癌的发生和发展密切相关,我国是乙型肝炎的高发区,在全球3.5亿HBV携带者中,约9300万,发展为慢性乙型肝炎的患者大约有2500万,其中每年约有1％的乙型肝炎患者发展为重型肝炎。重型肝炎(肝衰竭)可被分为四类：急性肝衰竭(acute liver failure, ALF)、亚急性肝衰竭(subacute liver failure, SALF)、慢加急性肝衰竭(acute-on-chronic liver failure, ACLF)和慢性肝衰竭(chronic liver failure, CLF)、慢加急性肝衰竭是在慢性肝病基础上出现的急性肝脏功能失代偿。在我们的第一部分实验中,我们已经发现慢性乙型肝炎患者外周血中Th1,Tc1细胞的比例明显低于正常对照。提示患者细胞免疫功能低下,不能有效清除体内的HBV,使HBV持续感染形成慢性化。Th17细胞亚群比例增加可能是一个重要的决定因素。在我国,慢性乙型病毒性肝炎是引起慢加急性肝衰竭的主要病因,有报道称CHB占ACLF所有病因的80％。由慢性乙型病毒性肝炎所致的慢加急性肝衰竭又称为慢加急性重型乙型病毒性肝炎(ACHBLF)。ACHBLF进展迅速,病情凶险,临床上缺乏特异、有效的治疗手段,绝大部分患者预后较差,易出现各种并发症而危及患者的生命,虽然近年来在ACHBLF的发病机制、临床诊断、治疗等方面取得了很大的进展,但死亡率仍高达50％-60％。目前,慢性乙型肝炎出现急性肝功能失代偿引起ACHBLF精确机制尚不明确,明确其发病机制必将提高ACHBLF的治疗水平,降低其死亡率。目的本研究的目的是通过对慢加急性乙型病毒性肝炎患者外周血Th17、Th1和Tc1细胞亚群及血浆细胞因子的检测,进一步探讨三种细胞亚群在慢加急性乙型病毒性肝炎患者发病机制中的作用。方法应用三色流式细胞术检测慢加急性乙型病毒性肝炎患者和正常对照外周血中Th1、Th17和Tc1细胞占CD3+T细胞的比例。三种细胞亚群的界定分别为：Th17细胞为CD3+CD8-IL-17A+,Th1细胞为CD3+CD8-IFN-γ+,Tc1细胞为CD3+CD8+IFN-γ+；应用ELISA技术检测慢加急性乙型病毒性肝炎患者和正常对照血浆中IL-17和IFN-γ的表达水平。结果1、慢加急性乙型病毒性肝炎患者外周血Th1细胞的比例(8.55±1.53％)明显低于正常对照组(10.56±1.91％)(P<0.01),并且慢加急性乙型病毒性肝炎患者Tc1细胞的比例(6.35±1.85％)较正常对照组(9.0±2.79％)亦显著升高(P<0.05)。2、慢加急性乙型病毒性肝炎患者Th17细胞的比例(1.53±0.52％)较正常对照(0.92±0.20％)显著升高(P<0.05)。3、在慢加急性乙型病毒性肝炎患者中,Th17与Th1细胞间亦存在显著的正相关性(r=0.61,P<0.05),但Th17与Tc1细胞间无相关性。4、慢加急性乙型病毒性肝炎患者血浆中IFN-γ和IL-17表达水平与正常对照相比均无显著性差异。结论我们的研究结果进一步表明,在慢加急性乙型病毒性肝炎的异常免疫反应中,Th1和Tc1型免疫反应占优势地位,并且Tc1细胞在慢加急性乙型病毒性肝炎的发病中可能发挥了细胞毒作用和免疫调节的双重作用。尤为重要的是,我们研究首次表明,在慢加急性乙型病毒性肝炎发生和发展中,Th17细胞亚群比例增加可能是一个重要的决定因素,其与Th1和Tc1细胞协同在慢加急性乙型病毒性肝炎的发生、发展中发挥了重要的作用,调节慢加急性乙型病毒性肝炎患者中Th17细胞的异常可能成为慢加急性乙型病毒性肝炎治疗的新策略。更多还原

【Abstract】 第一部分IMPLICATION OF TH17 AND TH1 CELLS IN PATIENTS WITH CHRONIC ACTIVE HEPATITIS BBackground Hepatitis B virus (HBV) infection is a major public health threat in the world, especially in China. Globally, there are approximately 350 million carriers of HBV, of whom 0.5-1.0 million die due to the HBV-associated liver disease each year. Both CD4+(Th) and CD8+(Tc) T lymphocytes can be functionally divided into type 1(T1) and type 2(T2) subsets based on the secretion of either IFN-γ(T1) or IL-4 (T2). Recent studies have provided compelling evidence for a third CD4+ T cell effector subset besides the well-described Th1 and Th2 CD4+ T cell. The newly identified CD4+ T effector cells have been named as Th17 cells. These cells are characterized as preferential producers of IL-17A (also known as IL-17), IL-17F, IL-21, IL-22, and IL-26 in humans. Retinoid orphan nuclear receptor (RORC), which encodes the human ortholog of mouse RORct, is a key regulator of Th17-cell lineage differentiation. Th17 cells and their effector cytokines are increasingly being recognized as key determinant in the induction of autoimmune diseases and malignant tumor as well as alcoholic liver disease. There are limited data to date about Th17 levels in patients with chronic hepatitis B (CHB).To further investigate the role of Th17 and Thl cells, as well as their relationship in the pathogenesis of CHB, we examined the frequency of Th17 and Thl cells in CHB active patients through intracellular cytokines analysis by flow cytometry, ELISA and real-time PCR.Objective The pathogenesis of hepatitis B virus (HBV) associated chronic liver disease is still not fully understood.The immune imbalance of cytokine profile exerts a profound influence on the resolution of HBV infections and HBV clearance. This present study aimed to evaluate the immune status of the peripheral T helper (Th) 17 and Th1 cells in the active patients with chronic HBV infection. Methods Thirty patients with chronic active hepatitis B were included in our present study. The frequency of peripheral Th 17 cells (CD3+CD8-IL-17+T cells), Thl cells (CD3+CD8-IFN-γ+T cells), and Tc1 cells (CD3+CD8+IFN-γ+T cells) in chronic hepatitis B(CHB) were analyzed by flow cytometry. The protein and mRNA levels of interleukin-17 (IL-17) and interferongamma (IFN-γ) were measured by enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction (PCR).Results The percentage of Th17 cells in peripheral blood of CHB patients (1.53±0.52%) was significantly increased than that in normal controls (0.92±0.20%; P<0.05). In contrast, the percentage of Th1 and Tc1 cells of CHB patients was significantly decreased as compared with that of control group. The frequency of Th17 cells had a negative correlation with Th1 cells, and a positive correlation with serum alanine aminotransferase in CHB patients.Conclusion Our study data strongly support a high Th1 and Tc1 polarization of the immune response in CHB and CTL-mediated cytotoxicity is an alternative mechanism in CHB. More important, our study demonstrates for the first time that Th17 may be an important determinant in the evolution of CHB along with Th1 and Tc1, suggesting that blocking the abnormality of Th17 cell in CHB is likely a promising therapeutic concept for CHB. 第二部分IMPLICATION OF TH17 AND TH1 CELLS IN PATIENTS WITH ACUTE-ON-CHRONIC HEPATITIS B VIRUS LIVER FAILUREBackground Hepatitis B virus (HBV) infection still poses a major public health threat in a large part of the world. Some CHB patients may rapidly progress towards liver failure, a condition referred to as acute-on-chronic liver failure(ACLF). In China, acute-on-chronic hepatitis B liver failure (ACHBLF) accounts for more than 80% of ACLF cases due to a high incidence of HBV infection. ACLF shares striking similarities with septic shock with regard to the features of systemic inflammation, progression to multiple organ dysfunction and functional immunoparesis.The precise mechanisms underlying the deterioration of liver function occurring in ACLF remain unclear.There are limited data to date about Th17 levels in patients with acute-on-chronic hepatitis B liver failure (ACHBLF).To further investigate the role of Th17 and Thl cells, as well as their relationship in the pathogenesis of ACHBLF, we examined the frequency of Th17 and Th1 cells in ACHBLF active patients through intracellular cytokines analysis by flow cytometry and ELISA.Objective Acute-on-chronic hepatitis B virus liver failure (ACHBLF) has been shown to carry poor prognosis, however, the pathogenesis of ACHBLF is still not fully understood. This present study aimed to evaluate the immune status of the peripheral T helper (Th) 17 and Thl cells in the active patients with ACHBLF.Methods Twenty patients with ACHBLF were included in our present study. We examined the levels of Th17,Th1 and Tc1 cells in peripheral blood which was activated in vitro by PMA/ionomycin in short-term cultures in ACHBLF patients and controls by flow cytometry through intracellular cytokines analysis. Th17 cells and Thl cells were identified as those that were CD3+CD8-IL-17A+and CD3+CD8-IFN-γ+, and Tc1 cells were those that were CD3+CD8+IFN-γ+. ELISA was used to detect the expression of Th1 and Th17 cytokines (IFN-γand IL-17) in plasma. Results The results showed that the percentages of both Thl and Tc1 cells in ACHBLF patients increased significantly compared with the normal controls (P<0.01 for Thl, P<0.05 for Tc1). More importantly, the percentage of Th17 in patients with ACHBLF was markedly higher than that of normal controls (P＜0.05). Also, the percentage of Th17 cells positively correlated with Thl cells (r=0.61, P＜0.05). There was no significant correlation between Th17 and Tc1 cells (r=0.09, P=0.71).Conclusion Our study demonstrates for the first time that Th17 cells may be an important determinant in the evolution of ACHBLF along with Thl cells, suggesting that blocking the abnormality of Th17 cell in ACHBLF is likely a promising therapeutic concept for ACHBLF.更多还原