【作者】 陈永艳；

【导师】 田志刚；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2007， 博士

【摘要】 乙型肝炎病毒（HBV）作为一种非细胞毒性的嗜肝病毒，感染机体后将导致急慢性肝炎、肝硬化、肝癌等一系列肝脏疾病的发生，成为危害人类健康的一项严重问题。机体对病毒抗原的免疫反应是HBV引起肝脏疾病的重要机制，但目前HBV确切的致病机理仍不清楚。随着肝脏天然免疫系统的提出，肝脏内天然免疫细胞在HBV感染致病过程中的作用引起了学者们的重视，尤其是肝脏NK细胞。肝脏内聚集着大量的NK细胞，约占肝脏淋巴细胞总数的35％。NK细胞在机体抗HBV感染中起着不可忽视的作用，但是在HBV感染肝脏免疫致病过程中的作用及其可能的机制还有待于进一步的研究证实。值得注意的是人类HBV健康携带者再受到外界因素如病毒、细菌等感染、化学物质、细胞刺激剂、妊娠及免疫治疗等作用后肝细胞极易发生损伤，严重危害这一人群的健康。有研究提示NK细胞参与HBV转基因小鼠肝脏易损伤过程，但是NK细胞通过何种机制参与肝细胞敏感损伤尚未阐明。故本研究利用HBV转基因小鼠C57BL／6J-TgN（AlblHBV）44Bri，一种模拟人类HBsAg健康携带者的模型，深入探讨肝脏天然免疫细胞在HBV感染肝脏免疫损伤中的作用及机理。首先分析比较HBV转基因小鼠中枢及外周免疫系统中各类淋巴细胞的存在状态，尤其是肝脏NK细胞的基本特性；利用多种刺激剂如Poly I：C，Con A及CCl₄研究HBV转基因小鼠肝脏损伤情况，并进一步探索HBV转基因小鼠肝脏NK细胞的免疫应答特性及在肝脏损伤中的作用。实验过程中，采用赖氏法检测血清转氨酶ALT／AST水平、组织H-E染色观察肝脏病理学变化，并利用上述指标综合评估肝细胞损伤程度；通过细胞清除和细胞过继转输技术，研究特定淋巴细胞群在肝脏损伤过程中的作用以及细胞间相互作用关系；采用流式细胞术测定各淋巴细胞亚群的表型，活化状态以及细胞因子分泌功能；利用Western blotting方法检测肝细胞胞内信号传导；运用特异抗体中和实验观察细胞间相互作用的分子机制；运用特异性抗体阻断实验以及体外AST释放实验探索淋巴细胞与肝细胞间相互作用机制。通过上述研究方案获得如下结果：1．HBV转基因小鼠肝脏NK细胞特性改变与正常野生型小鼠相比，HBV转基因小鼠肝脏NK细胞数量明显减少；自然活化程度（CD69表达水平）明显下降；对肿瘤细胞的细胞毒活性明显受损；表面TRAIL分子的表达水平明显下降。而且，HBV转基因小鼠肝脏NK细胞对Poly I：C的免疫应答不同于正常野生型小鼠。虽然Poly I：C能够刺激HBV转基因小鼠肝脏NK细胞的聚集、活化、并提高其对肿瘤细胞的细胞毒活性，但是HBV转基因小鼠活化的肝脏NK细胞细胞毒活性仍明显低于正常野生型小鼠。2．HBV转基因小鼠肝脏损伤敏感性明显增加低剂量Poly I：C（3μg／g body weight）诱导HBV转基因小鼠血清转氨酶ALT／AST急剧升高，严重的肝细胞坏死和肝脏炎性浸润，与高剂量Poly I：C（30μg／g body weight）的作用基本相同；而在正常野生型小鼠，仅高剂量Poly I：C的作用引起血清转氨酶ALT／AST的轻度上升。低剂量Con A（3μg／g body weight）刺激下，正常野生型小鼠血清转氨酶ALT／AST仍在正常范围内；而HBV转基因小鼠血清转氨酶ALT／AST急剧升高，肝脏有明显的大片坏死灶及炎性浸润。足剂量Con A（15μg／g body weight）作用后HBV转基因小鼠死亡率明显高于正常野生型小鼠。另外，HBV转基因小鼠对于化学毒物CCl₄诱导的肝细胞损伤敏感性也显著增加。刺激24h后HBV转基因小鼠血清转氨酶ALT急剧上升，显著高于正常野生型小鼠。3．HBV转基因小鼠对Poly I：C诱导的肝脏损伤敏感性增加的机制：NK细胞及IFN-γ依赖途径细胞清除实验表明HBV转基因小鼠中，低剂量Poly I：C（3μg／g body weight）诱导的肝细胞敏感损伤依赖于NK细胞。低剂量Poly I：C刺激后，HBV转基因小鼠肝脏内活化的NK细胞分泌大量IFN-γ。中和体内IFN-γ后，低剂量Poly I：C诱导的肝脏敏感损伤明显减弱。给予外源性重组IFN-γ后血清转氨酶ALT／AST水平表明HBV转基因小鼠对IFN-γ诱导的肝细胞损伤作用更为敏感。进一步研究发现HBV转基因小鼠肝细胞表面IFN-γ受体的表达水平明显高于正常野生型小鼠，而且低剂量Poly I：C刺激明显提高其表达。在IFN-γ刺激后HBV转基因小鼠肝细胞胞内信号pSTAT1-IRF-1明显强于正常野生型小鼠肝细胞。另外，在HBV转基因小鼠Poly I：C诱导的肝脏敏感损伤过程中，NK细胞发挥作用不依赖于Kupffer细胞及IL-12。4．HBV转基因小鼠对Con A诱导肝脏损伤极度敏感的机制：NKG2D依赖途径细胞转输实验证明在低剂量Con A（3μg／g body weight）诱导肝细胞损伤过程中，与正常野生型小鼠相比HBV转基因小鼠肝脏淋巴细胞对肝细胞的杀伤活性明显增强，同时HBV转基因小鼠的肝细胞对淋巴细胞的杀伤作用更为敏感。低剂量ConA作用下，HBV转基因小鼠肝脏内聚集着更多的活化NK细胞，而且低剂量Con A诱导的肝脏敏感损伤依赖于NK细胞。纯化的NK细胞体内转输实验证明低剂量Con A活化的肝脏NK细胞具有肝细胞毒活性，是肝细胞损伤的效应细胞。Con A刺激后HBV转基因小鼠肝细胞不同于正常野生型小鼠肝细胞，其NKG2D配体（Rae-1和Mult-1）的表达明显上调。阻断HBV转基因小鼠体内NKG2D通路后，低剂量Con A诱导的肝细胞损伤明显减弱。体外纯化的NK细胞对肝细胞的杀伤实验（4-h AST释放实验）进一步证明低剂量Con A活化的NK细胞对肝细胞的细胞毒作用依赖于NK细胞与肝细胞之间NKG2D-ligand相互识别。另外，在低剂量Con A诱导HBV转基因小鼠肝脏敏感损伤过程中，活化的NKT细胞通过分泌细胞因子IFN-γ和IL-4对NK细胞活化发挥重要的正向调节作用。5．HBV下调人肝癌细胞系MHC-Ⅰ类分子的表达利用转染HBV的HepG2．2．15细胞系及其对照细胞系HepG2，发现HBV感染显著抑制肝癌细胞表面HLA-ABC分子的表达，HLA-E mRNA的表达；显著下调肝癌细胞膜型MICA分子及MICA mRNA的表达。本研究首次揭示了HBV转基因小鼠肝脏NK细胞的特性及HBV感染肝脏免疫损伤高度敏感的天然免疫机制，同时对HBV感染对人肝细胞MHC-Ⅰ类分子表达的影响进行了初步的探讨。本研究将为揭示人类HBV慢性携带者肝脏NK细胞的基本特性及肝细胞损伤敏感性增加的免疫学机理提供重要依据，对控制、治疗HBV慢性感染导致的多种肝脏疾病具有一定的指导意义。更多还原

【Abstract】 Hepatitis B virus （HBV） as a hepatotropic, noncytopathic virus, primarily infects hepatocytes and causes a series of liver diseases such as acute and chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, which are among the most important human health problems worldwide. It has been reported that the immune responses are fundamental for viral clearance and pathogenesis during HBV infection; however, the precise pathogenesis has not been well known until now. With the new recognition that liver is a lymphoid organ with an overwhelming innate immune system, the roles of innate immune cells during HBV infection, especially the hepatic NK cells, deserve further investigating. NK cells are abundant in the normal liver, accounting for about one third of intrahepatic lymphocytes. Available evidences have indicated that hepatic NK cells play an important anti-viral role during HBV infection, but the role in the development of HBV-associated liver injury remains obscure. Noticeably, human healthy HBsAg carriers are susceptible to hepatocyte injury induced by a variety of elements such as stresses, infections, pregnancy and use or withdrawal of immunosuppressive therapies, which is a serious risk for the health of chronic HBsAg carriers. The basis for the susceptibility has not been understood yet. It has been argued that NK cells were involved in the over-sensitive liver injury in HBV transgenic mice triggered byα-GalCer, but the actual mechanisms of NK cells in hepatocyte injury remain elusive.In this study, we used HBV transgenic mice C57BL/6J-TgN （AlblHBV） 44Bri, which mimic human healthy chronic HBsAg carriers, to determine the functions of hepatic NK cells and the precise roles of innate immune responses in HBV-associated liver injury. The lymphocytes of central and peripheral immune organs were compared between HBV transgenic mice and wild C57BL/6 mice, especially hepatic NK cells. Poly I:C, Con A and CCl₄ were used to induce liver injury of HBV transgenic mice, and then immune responses of hepatic NK cells and precise roles of NK cells in the liver injury were investigated further. We examined the liver injury by determining serum transaminase ALT/AST level and liver pathologic changes. By cell depletion and cell transfer experiments, effects of the specific lymphocyte population and cellular interactions in the process of liver injury were investigated. The FACS analysis provided us with the precise information about lymphocyte phenotype, activation and cytokine production. Western blotting was applied to determine signals in heptocytes. Moreover, specific neutralization with antibody was applied to explore the potential mechanisms involved in cell interaction. Blocking with antibody and 4-h AST release experiments were applied to explore the molecular interaction between lymphocytes and heptocytes. Our major findings are shown as followed:1. Impaired function of hepatic NK cells from murine chronic HBsAg carriersCompared with wild C57BL/6 mice, in HBV transgenic mice the number of hepatic NK cells was decreased; natural activation （CD69 expression） of hepatic NK cells was declined; and cytotoxicity of hepatic NK cells was attenuated, which might relate to the down-regulated expression of TRAIL on hepatic NK cells. Additionally, although in the liver of HBV transgenic mice NK cells could be accumulated and activated by Poly I:C injection, the increase in anti-tumor cytotoxic activity of intrahepatic activated NK cells was markedly impaired in HBV transgenic mice compared with that in wild C57BL/6 mice.2. Murine chronic HBsAg carriers were over-sensitive to liver injuryA much low dose of Poly I:C （3μg/g body weight） injection induced much higher elevations of serum ALT and AST, severe hepatocyte necrosis and inflammation in HBV transgenic mice, which was similar to a high dose of Poly I:C （30μg/g body weight） injection. However, in wild C57BL/6 mice, there were only slight elevations of serum ALT and AST after 30μg/g body weight Poly I:C injection. These results demonstrated that HBV transgenic mice were over-sensitive to Poly I:C-induced liver injury.The nonhepatotoxic low dose of Con A （3μg/g body weight） for wild C57BL/6 mice induced severe liver injury in HBV transgenic mice, demonstrated by high level of serum ALT/AST and liver massive necrosis and inflammation. High dose of Con A （15μg/g body weight） injection induced the peak level of serum ALT/AST hardly with death in wild C57BL/6 mice, but caused death of most HBV transgenic mice.CCl₄ injection also induced much more severe hepatocyte injury in HBV transgenic mice than that of wild C57BL/6 mice shown by the level of serum ALT after 24 h of injection.3. Murine chronic HBsAg carriers were over-sensitive to Poly I:C-induced liver injury in NK cell- and IFN-γ-dependent mannerPoly I:C-induced over-sensitive liver injury was absolutely dependent on the presence of NK cells in HBV transgenic mice. After Poly I:C injection, intrahepatic NK cells were activated and produced IFN-γ. Neutralization of endogenous IFN-γsignificantly inhibited the over-sensitive liver injury. HBV transgenic mice were hypersensitive to IFN-γin liver injury, indicated by the results of serum ALT/AST after recombinant mIFN-γadministration. Further, much stronger IFN-γreceptor expression was observed on hepatocytes of HBV transgenic mice, which was significantly enhanced by Poly I:C injection. Treatment with IFN-γin vitro triggered much higher activation of downstream signals （pSTAT1-IRF-1） in hepatocytes of HBV transgenic mice. Depletion of Kupffer cells and neutralization of endogenous IL-12 did not affect Poly I:C-induced over-sensitive liver injury in HBV transgenic mice.4. Involvement of NKG2D activation in Con A-induced over-sensitive liver injury of murine chronic HBsAg carriersAfter low dose of Con A stimulation, hepatic MNCs became more cytotoxic and hepatocytes more sensitive to cytotoxicity from HBV transgenic mice compared with wild C57BL/6 mice. Low dose of Con A-induced over-sensitive liver injury was dependent on the more accumulated intraheptic NK cells in HBV transgenic mice, demonstrated by cell depletion. Adoptive transfer of purified hepatic NK cells from low dose of Con A-treated HBV transgenic mice further confirmed activated intrahepatic NK cells were cytotoxic to hepatocytes as effectors. Upon stimulation of low dose of Con A, expressions of NKG2D ligands （Rae-1 and Mult-1） in hepatocytes of HBV transgenic mice were markedly enhanced, but not in wild C57BL/6 mice. NKG2D activation of NK cells via recognition with Rae-1 or Mult-1 on hepatocytes accounted for the over-sensitive hepatocyte injury in HBV transgenic mice. Interestingly, NKT cells triggered by low dose of Con A served as regulator necessary forNK cell activation via secreting IFN-γand IL-4. 5. HBV down-regulated expressions of MHC class I molecules on hepatoplastoma cell lineIt was found that the expressions of HLA-ABC protein, HLA-E mRNA, membrane MICA protein and MICA mRNA were much lower in HepG2.2.15 cells compared with HepG2 cells. The expressing HBV in human hepatoplastoma cell line significantly down-regulated the expressions of MHC class 1 molecules.Here, we for the first time demonstrated the characterization of hepatic NK cells and revealed the precise roles of NK cells in the over-sensitive liver injury of HBV transgenic mice. Additionally, the possible effect of HBV on MHC class I molecules in human hepatocytes was investigated. These findings would be helpful to interpret the immune responses of hepatic NK cells and the immunological mechanisms of hepatocyte susceptibility to injury in human chronic HBsAg carriers.更多还原