於FVB/N小鼠建立B型肝炎病毒慢性帶原模式以研究宿主與病毒的影響因素

Abstract

研究B型肝炎病毒（hepatitis B virus，HBV）造成慢性帶原的機制一直是個很有挑戰性的問題。由於缺乏適合之HBV長期帶原的動物模式，因此感染病毒之後如何轉變成慢性帶原的成因仍然不清楚。在本研究中，我們利用高壓流體注射法（hydrodynamic injection）將病毒replicon DNA打到BALB/c、C57BL/6、FVB/N三種不同品系的小鼠身上，該replicon DNA便能在肝臟複製並產生病毒顆粒。在BALB/c、C57BL/6小鼠可以很快的將病毒清掉，然而我們發現在FVB/N小鼠身上HBV可以帶原長達五十週之久。以流式細胞儀與反轉錄定量PCR分析小鼠的肝臟發現FVB/N小鼠的毒殺性T細胞（cytotoxic T lymphocyte，CTL）、血清轉氨酶（alanine aminotransferase，ALT）、丙型干擾素（interferon-γ，IFN-γ）、甲型腫瘤壞死因子（tumor necrosis factor-α，TNF-α）、第九、第十號趨化激素（CXCL9、CXCL10）比另外兩種品系小鼠產生得少，反映出肝臟發炎程度與病毒清除效率的相關性。利用突變分析進一步證明若將HBV表面抗原的第214個胺基酸由Asparagine轉變成Serine則會使原本能夠帶原的clone被清除掉，此現象亦伴隨著活化的CTL、IFN-γ、CXCL9、CXCL10的增加。此動物模式亦適合用來評估抗HBV藥物的療效。在帶原小鼠增加CXCL9、CXCL10與補體5a（Complement 5a，C5a）的表現能夠促進病毒的清除。本研究的結果指出，不同的宿主遺傳背景與病毒序列會影響對抗HBV的免疫反應。免疫系統或發炎反應不恰當的活化可能導致HBV的慢性帶原。

The mechanism underlying the chronicity of hepatitis B virus (HBV) infection has long eluded researchers. The mechanism has remained unclear largely due to the lack of an animal model that can support persistent HBV replication and allow for the investigation of the relevant immune responses. In this study, we used hydrodynamic injection to introduce HBV replicon DNA into the livers of three different mouse strains, BALB/c, C57BL/6, and FVB/N. Interestingly, we found that an HBV clone persistently replicated in the livers of FVB/N mice for up to 50 weeks but was rapidly cleared from the livers of BALB/c and C57BL/6 mice. Flow cytometric analysis and quantitative reverse transcription PCR analysis of the mouse livers indicated that after DNA injection, FVB/N mice had few intrahepatic activated cytotoxic T lymphocytes (CTLs) and produced low levels of alanine aminotransferase, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and the CXCL9 and CXCL10 chemokines. These responses were in sharp contrast to those observed in BALB/c and C57BL/6 mice, reflecting a strong correlation between the degree of liver inflammation and viral clearance. Mutational analysis further demonstrated that a change from Asn-214 to Ser-214 in the HBV surface antigen permitted the clearance of the persistent HBV clone in FVB/N mice, and the response was accompanied by increased levels of activated CTLs and upregulated liver expression of IFN-γ, CXCL9, and CXCL10. The model was demonstrated to be useful for the in vivo evaluation of the efficacies of various anti-HBV drugs. Supplementary expression of CXCL9, CXCL10 and C5a in the carrier mice enhanced the clearance of the chronic infection. These results indicate that the heterogeneity of the host factors and viral sequences may influence the immune responses against HBV. An inadequate activation of immune or inflammatory responses can lead to persistent HBV replication in vivo.