The Establishment Of A HCV Mouse Model And Its Application In The Study Of Immune-mediated Liver Injury

Hepatitis C virus (HCV) is a single strand hepatotropic RNA virus of flavivirus, and is one of the major causative factors leading to the chronic hepatitis. Clinical epidemiology data show that susceptibility to liver injury represents a serious problem for the huge population of chronic HCV carriers. And an increased risk for progression to cirrhosis and hepatocellular carcinoma. However, no vaccine or useful treatment against HCV infection is currently available. So the studies that forcus on the basic research on Hepatitis C virus should be strengthened.HCV replicates in hepatocytes but is believed to be non-cytopathic. Also, previous studies have indicated that liver injury secondary to HCV infection is likely immune-mediated and does not result from direct cytopathic effects of the virus. The immune system, especially the intrahepatic immune system are thought to cause the associated liver damage. However, the roles of hepatic innate immune cells in the liver pathogenesis of chronic HCV infection remain poorly understood. Therefore, the aim of this study was to explore the interaction between HCV and host immune cells. There are two parts in this study:1. The exploration and improvement of liver-specific transfection technologyA suitable animal model is important for the study of the interaction between HCV and host immune cells. The animal models which could infect HCV is limited to ’chimpanzees, various transgenic and chimeric transgenic mice transplanted with’ human hepatocytes mouse model. Factors that limit the use of these animal models, including ethical concerns, restricted availability and high cost. The HCV transgenic mouse model is useful in the studies of interaction between HCV proteins and host. But this mouse model is immunotolerant to HCV, making it difficult to study the interaction between HCV and host immune system. Therefore, a new suitable animal model that can overcome these limitations is needed.1.1The establishment of the retro-orbital sinus hydrodynamic-based transfectionWe developed the retro-orbital sinus hydrodynamic injection base on the hydrodynamic transfection technique and retro-orbital sinus injection. And this technique enable the expression of exogenous gene especially in liver. The transfection efficiency of retro-orbital sinus hydrodynamic injection are same to the traditional hydrodynamics-based technique. And the retro-orbital sinus hydrodynamic injection is more simple and convenient for operation. The main contribution of this work is the establishment of an alternative route of DNA injection for hydrodynamic gene delivery, especially for those animals whose tail veins are not visible. And these effective expanding the applications of the hydrodynamic transfection technology.1.2Stable integration of foreign gene in murine livers by the mouse codon-optimizedφC31integraseA previous study demonstrated that the cpC31integrase system represents a potential modality for genetic engineering of the mouse genome. Recently, a mouse codon-optimized cpC31integrase, namely cpC31o, which greatly improved the recombination efficiency in the mouse genome, facilitated a potentially broader range of molecular manipulations.In order to promote the long-term expression of foreign gene in mouse liver, the cpC31o integrase technology is introduced in this study. We established a method for stable transfection of the hepatic tissues and applied the transfected mice to longitudinal monitoring of NF-κB activity under pathological conditions. Compared with the non-codon-optimized φC31integrase, the new system are more effective in the integration of the foreign gene to mouse liver chromosome.1.3The establishment of HCV expression mouse model based on the hydrodynamic transfection and φC31o integraseBased on the hydrodynamic transfection technology and the codon-optimized cpC31integrase technology, we established a long-term HCV expression animal model in the immunocompetent C57BL/6mice. Furthermore, the use of luciferase as the reporter for HCV made it possible to monitor the expression of HCV in mouse liver by in vivo imaging in real time. 2. The study of HCV expression renders hypersensitivity to ConA-induced liver injuryNK cells participate in innate immune responses that provide efficient direct antitumor and antiviral defenses. Over the years, circumstantial evidence has suggested that the activation of hepatic NK cells represents a pivotal factor in the pathogenetic processes, leading to progressive tissue injury and ultimately to cirrhosis. However, the role of the liver NK cells in pathogenetic processes leading to liver injury remains poorly understood.2.1The influence of HCV expression on the liver immune cellsBased on the immunocompetent character, this model can be used to explore the interaction between HCV and host immune cells. We analyzed the influence of HCV expression on the liver immune system. The results showed that the number of liver NK cells in HCV mouse was reduced, but did not change the activity of hepatic NK cells. And the other types of hepatic lymphocytes exhibited no difference (NKT cells, T cells and DC cells). These results are consistent with the clinical reports.2.2HCV expression renders hypersensitivity to ConA-induced liver injuryConsidering the important role of NK cells in the pathogenesis of HCV-induced hepatitis. This mouse model was used to examine the regulatory effects of HCV on immune-mediated liver injury. Autoimmune hepatitis was induced by a systemic injection of ConA. The results show that the injection of ConA induced significantly more severe hepatocyte injury in HCV-expressing mice compared to control mice. Following ConA injection, activation of NK cells was increased in livers with integrated HCV, and the depletion of NK cells elicited liver-protective effects against ConA-induced liver damage. In addition, our results indicated that the increased production of perforin, IFN-γ and TNF-α and the NKG2D/NKG2D ligand signaling pathway played a crucial role in NK cell-mediated liver injury in HCV mice after the administration of ConA.In summary, this study described an immunocompetent HCV mouse model combined with the hydrodynamic transfection technique, the mouse-codon-optimized φpC31integrase and the in vivo imaging. Our study showed that this mouse model is more sensitive to ConA-induced liver injury, and this is closely associated with abnormal activation of NK cells in the liver. And the expression of NKG2D and the production of perforin, IFN-y and TNF-a by hepatic NK cells play the major roles in pathologic immune injury in the liver of HCV mouse. And the hepatic NK cells are greatly activated via NKG2D/H60interaction. As this mechanism is important in the context of human liver diseases, this HCV mouse model represents an interesting tool that offers insight into HCV-induced liver pathophysiology and can potentially facilitate the development of future therapeutic strategies.