【作者】 王洪；

【导师】 王宇明；

【作者基本信息】 第三军医大学 ， 内科学， 2004， 博士

【摘要】 乙型肝炎病毒（hepatitis B virus,HBV）感染仍为全球重要健康问题之一,大约有3亿5千万慢性感染人口,每年死于HBV感染的人数达1百万。我国HBV感染人群比例更大,估计占总人口的10%。HBV感染可出现多种临床表现,包括急性、慢性、重型肝炎,可发展为肝硬化、肝癌,最终常因肝衰竭而死亡。上述复杂疾病谱的发病机制尚不清楚,目前一致认为其发病机制中免疫反应占主导地位,受MHC-Ⅰ类分子严格限制的抗原特异性CTL识别感染肝细胞是造成肝损伤的主要原因,抗原特异性CTL在HBV感染发病中具有重要作用。 对HBV感染免疫发病机制的研究发现,HBV感染时抗原特异性CTL反应很弱,特别是慢性HBV感染,用传统检测抗原特异性CTL的方法,如有限稀释法（limiting dilution assays,LDA）、⁵¹Cr释放试验法、酶联免疫斑点法等方法几乎检测不到,这种情况严重阻碍了HBV感染抗原特异性CTL的研究。自从1996年Altman等发明了MHC-表位肽四聚体技术以来,四聚体技术已经广泛用于基础和临床研究,也推动了HBV感染抗原特异性CTL的研究。四聚体技术直接检测抗原特异性CTL,无需体外扩增,是一种高效敏感的方法。 四聚体复合物是用生物技术将MHC-Ⅰ分子的C末端接一个生物素化位点,然后与特异性表位肽结合为MHC-Ⅰ抗原肽复合物,再按照4∶1的比例结合在亲和素上成为MHC-Ⅰ四聚体复合物。四聚体复合物可同时识别针对这段表位肽的四个T细胞受体（T cell antigen receptor,TCR）,与T细胞紧密结合,从而识别特异性CTL。四聚体技术具有很强的通用性,即相同的MHC表型个体之间,四聚体复合物可以通用,无需考虑T细胞受体的多样性。另一方面,四聚体复合物结构简单,对适用于相同MHC表型个体的各种四聚体复合物之间的差异仅在所采用的表位肽。因此,构建一个MHC-表位肽四聚体也就方便了其他肽特异性四聚体复合物的构建。 四聚体技术用于HBV感染抗原特异性CTL的研究已有几年,但多限于核心抗原18-27、衣壳抗原335-343和多聚酶抗原575-583,而且所采用的病例数较少,难以反映HBV感染抗原特异性CTL的真实状态。因此,更多病例,更多抗原表位肽特异性更多还原

【Abstract】 Hepatitis B virus (HBV) may cause both acute and chronic infection. Chronic infection is the major causes of chronic liver inflammation, leading to cirrhosis, hepatic decompensation, and hepatocellular carcinoma. The pathogenesis of HBV infections is not well understood, most studies indicate that HBV is not cytopathic for the infected hepatocyte, the cellular immune response is thought to be critical in determining the outcome of infection in terms of both viral clearance and liver cell injury. Therefore, understanding the antigen-specific immune response against HBV is important for the development of successful therapy and vaccine for this serious public health problem.The antigen-specific T cells in patients with HBV infection, especially the chronic infection, is so little that it can not be indetected by conventional methods, such as chromium-release assays, limiting dilution assays (LDA), and enzyme-linked immunosorbent spot assay (ELISPOT). Study on the specific CTL had long been hampered by lack of suitable technques. Since Altman published the first paper in 1996 describing the use of major histocompatibility complex (MHC) class peptide tetrameric technology for direct visualization and quantification of antigen-specific cytotoxic T cells, there have been many tetramer-based studies in both basic and clinical immunology. The tetrameric technology is a powerful tool in researching on antigen-specific T cells, regardless of their function after in vivo or in vitro antigen stimulation, and without the need of in vtro expansion, it have greatly enhanced the opportunities for directly studying antigen-specific T cells.The tetrameric technology involves the engineering of a biotinylation sigal sequence onto the C terminus of a recombinant MHC class I molecule which, after complexing with a specific peptide is bound to avidin at a 4:1 ratio. This results in a tetrameric peptide-MHC complex that recognizes T cell receptors (TCRs) on lymphocytes specific for the particular epitope, the particular peptide determines the specificity of tetramer. So, ifyou can synthesis one tetramer, it is easy for other one.Although the tetrameric technology was used in the research of HBV infection for several years, but a few epitopes and a few cases of patients with HBV infection were involved, these results were not enough confidenty. In order to study extensively on the HBV-specific CTL using tetramers, we tried to construct the HBV-peptide tetramer, and we used four HLA-A2 tetramers, specific for HBV core (18-27), HBV envelope (183-191), (335-343) and polymerase (575-583), to investigate the frequency of epitope-specific CTL in PBMCs of chronical hepatitis B, to evaluate the correlation between the specific T cells and the outcome of infectionThe main research results are as follows:1. The construction of HBV-peptide tetramer.Upon addition of IPTG, the recombinant HLA-A2-BSP and P -2 microglobulin was produced in E.coli BL21(DE3)pLysS transformed by the expression plasmid. The 6x His-tagged fusion protein was purified using Nickel Chelated Agarose, HLA-A2-BSP was folded in the presence of 0 -2M and a HBV peptide to form a peptide-MHC complex, and the MHC complex were bitotinylated using BirA enzyme. Tetramer were generated by mixing bitotinylated protein complex with streptavidin-PE at a molar ratio of 4:1.2. Establish the method of using MHC- peptide tetramer in detecting antigen- specific CTLs.HLA-A2+ HBV-negative blood donors and HLA-A2- negative patients with chronic hepatitis B were tested as negative controls, we used the tetramers to test peripheral blood from the HLA-A2+ patients with chronic HBV infection and controls. The frequency of tetramer- positive T cells exceeded 0.02% circulating CD8+ cells which represent the maximum staining observed in the controls, so the cutoff for tetramer-positive was set at 0.02%. The frequency of tetramer- positive T cells in the A2+ chronic HBV infection was 0-2.41%.3. Research on the frequency of antigen- specific T cells in the peripheral blood from patients with hepatitis B.The frequency of HBV-specific CTL in the circulation was determined by the tetramer in 2 individuals with self-limited HBV infection, 36 chronically infection patients and 5 serious hepatitis. The frequencyof of tetramer-positive T cells was 0-2.41% in patients withHBV infection, numbers of tetramer- positive cells was above the control level, and more than one epitope specific CTL in 34 out of 36 chronic patients was demonstrated. But the positive rate of whole 4 tetramer cells was only higher in two patients with self-limitited HBV infection. Although tetramer-positive cells specific for one peptide (especially envelope 183-191) were found at a high frequency in a few chronically infection patients, the frequency of tetramer- positive T cells was low in most of these patients. The results were coincidence with that by intracellular staining and ELISPOT for Y -IFN. The number of tetramer binding cells was not different in chronic infection and serious hepatitis.4. Research on the antigen- specific T cells in the peripheral blood from patients with hepatic flares36 chronically infection patients were divided into four groups according to the levels of serum alanine aminotransferase (ALT). 1st group: ALT< 1 x ULN(upper limit of normal), 2nd group: ALT^2xULN, <5xULN, 3rd group: ALT2?5xULN, <10xULN, 4th group: ALT^lOxULN. We studied the number and function of the specific cytotoxic T lymphocytes in each group. The results indicated that frequency of three epitope specific CTL (C18-27, El83-191 and P575-583) was higher in 3rd group.6 patients who had elevated serum alanine aminotransferase (ALT) were monitored for 9 monthes, the frequency of tetramer- positive T cells was reduced as the ALT descended in 3 patients. In one patients, the number of tetramer- positive T cells was fluctuated following the changes of ALT.Conclusion:1. Purified the recombinant protein of HLA-A2-BSP and 0-2 microglobulin, and completed the construction of one HBV peptide tetramer.2. The tetrameric technology is a specific tool in enumerating antigen-specific T cells, the HBV- HLA-A2 tetramers was only used in HLA-A2-positive HBV infections.3. The frequency of tetramer-positive cells was different between the self-limited HBV infection and chronically infection. Self-limited HBV infection mount a multispecific polyclonal CTL response to several HBV-antigens. In contrast, the HBV-specific immune response is weak in the blood of chronically infected patients.4. Virus and antigen specific CTLs coexist in the chronic HBV infection. The presence of HBV-specific T cells are not determinant of immune-mediated protection inHBV infection.5. The frequency of HBV-specific T cells was highly when hepatitic flares occur. It indicated that the specific cellular immune was boost up, and the HBV-specific T cells may contribute to liver injury in chronic infection.更多还原