【作者】 范荣；

【导师】 王福生；

【作者基本信息】 中国人民解放军军医进修学院 ， 传染病学， 2010， 博士

【摘要】 乙型肝炎病毒（HBV）是一种嗜肝性DNA病毒,病毒本身的复制并不造成肝脏损伤。研究表明,CHB患者肝脏损伤是由于免疫系统不能有效控制病毒复制,却又长期被激活并介导肝细胞破坏而发生的。近十年来,人们逐步开始认识到NK细胞在各类肝脏疾病发生发展过程中发挥了重要作用。然而,HBV慢性感染患者（特别是e抗原阳性的CHB患者）体内NK细胞的功能特点及其临床意义还未被揭示,NK细胞介导的肝损伤机制也未完全阐明。本研究通过横断面研究,系统观察HBV感染各个时期（包括急性肝炎、慢性HBV感染免疫耐受期和免疫活化期）患者外周血NK细胞的频率、表型和功能特点,并密切结合患者的临床指标（谷丙转氨酶、谷草转氨酶水平和血清HBV载量）进行分析,以期阐明NK细胞变化的临床意义,并在此基础上初步探讨了CHB患者NK细胞免疫活化的可能原因及其介导肝脏免疫损伤的作用机制。研究发现,与免疫耐受期患者和健康对照者比较,慢性乙肝免疫活化期患者外周血NK细胞亚群发生显著改变,表现为CD56^dimCD16^pos亚群频率下降而CD56^brightCD16^neg亚群频率上升。我们还发现尽管急性乙型肝炎病程早期阶段,患者的外周血T细胞和NK细胞表面的活化标志物如HLA-DR、CD38和CD69的表达显著上升,然而慢性乙肝免疫活化期患者的外周血仅NK细胞活化标志显著增加,提示慢乙肝免疫活化期以NK细胞活化为主。进一步的体外功能试验揭示免疫活化期患者NK细胞在IL-12／IL-15刺激后表达更多IFN-γK562刺激后产生更多CD107a,同时杀伤更多的靶细胞K562、HepG2、HepG2.2.15和HuH7.5细胞,更重要的是NK细胞杀伤活性与肝损伤程度密切相关。本研究通过对乙型肝炎患者NK细胞的表型和功能进行详细分析,发现慢性乙型肝炎免疫活化期患者外周血NK细胞处于高活化状态,进一步支持慢性乙型肝炎患者NK细胞的高度活化与肝损伤的严重程度密切相关。更多还原

【Abstract】 Hepatitis B virus （HBV） is a hepadnavirus DNA virus, which does not directly cause liver damage. Therefore, liver damage in CHB patients is believed to be mediated by host immune responses. On one hand, inefficient control of viral replication occurs because specific anti-HBV immune response is inefficiently activated; on the other hand, non specific immune cells are persistently activated in a long period and will eventually mediate liver damages. Over the past decade, NK cells are gradually regarded as a very important player in the immunopathogenesis of various types of liver diseases. However, little is known about their immunological characteristics （including their phenotype and function details, especially during HBeAg-positive hepatic flare） as well as the immune-pathological mechanisms mediated by these cells.In present study, we conducted a comprehensive cross-sectional analysis of the immunological features （phenotype and functional characteristics） of peripheral NK cells in different phases of HBV-infected patients, including acute hepatitis, chronic HBV infection in immune tolerant phase and in immune activation phase. In order to explain the clinical significance of immunological changes and investigate mechanisms of NK cells activation and immune-mediated liver injury in CHB patients, clinical parameters （ALT, AST and viral load of HBV in serum） were also analyzed and correlated to these immunological features.We found that immune activated （IA） CHB patients displayed a large redistribution of peripheral NK cell subsets, indicated by decreased CD56^dimCD16^pos and increased CD56^brightCD16^neg subsets. More importantly, NK cells expressed significantly high levels of activation markers such as HLA-DR, CD38 and CD69 in IA patients rather than IT patients with chronic HBV infection. Interestingly, these activation molecules are simultaneously up-regulated on both T cells and NK cells in the patients at the early phase of acute resolved hepatitis B, which indicate that only NK cells are preferentially activated in IA patients. Functional analyses revealed that activated NK cells in IA patients secreted more cytokine IFN-y stimulated by IL-12/IL-15 and more CD 107a stimulated by K562 than IT patients and healthy controls. Also activated NK cells in IA patients could kill more target cells, including K562, HepG2, HepG2.2.15 and HuH7.5 cells, In addition, this hyper-activity of NK cells in IA patients was found to closely correlate with the higher level of serum ALT/AST.Our findings indicate that peripheral NK cells were highly activated in IA patients with CHB rather than IT subjects. Importantly, our data suggest that the over activities of NK cells are closely associated with the severity of liver damage in the CHB patients.更多还原