USP18和ISG20在α干扰素抗乙肝病毒机制中的作用及调控研究

Interferon alpha (IFN-α) has become one of the best available treatment options for chronic hepatitis B (CHB) patients. Due to the limitation of IFN-α treatment, such as low efficacy and high cost, it is critical to develop a prognostic tool to predict treatment response and to explore the underlying mechanism of treatment non-response. In our previous work, using microarray gene expression profiling, we identified a subset of differentially expressed genes in the liver tissues of treatment responders and non-responders. A lot of these genes are interferon-stimulated gene (ISGs) and immune-related genes. ISGs were upregulated significantly in non-responsive group, which is similar to the findings reported in chronic hepatitis C infected patients prior to IFN treatment. Most recently, some studies indicated that ISGs play essential role in activation of interferon-signaling pathway, and are closely related to the efficacy of IFN-α treatment. In addition, the differentially expressed ISGs may have predictive value for the efficacy of IFN-α treatment. As such, further study on the function and mechanism of ISGs is warranted. In this current study, we intend to focus on the key points of these differentially expressed genes and to explore their functions and mechanisms in regulating IFN signaling and anti-viral effect of IFN. USP18 and ISG20, two key ISGs identified from our previous study, will be modulated to study the function and mechanism in IFN-α non-response in CHB patients. Our study will be performed on multiple levels, such as mRNA and protein level. Our research will likely provide novel therapies for CHB through understanding of the underlying mechanism of treatment non-response.

慢性乙型肝炎是我国的常见病，其危害大，容易发生肝硬化、肝癌等不良后果。α干扰素抗病毒治疗是目前公认的有效治疗方法之一，但部分患者治疗效果差，加之价格昂贵，在很大程度上限制了其在临床的广泛使用。因此，积极探索α干扰素抗HBV机制、增加其治疗有效率是临床亟待解决的难题。我们在前期工作中，采用基因芯片技术筛选出ISGs中两个关键基因USP18和ISG20，发现二者与α干扰素抗HBV治疗效果密切相关。基于此，我们拟构建USP18、ISG20慢病毒载体，采用RNA干扰及JAK/STAT、NF-KB信号通路抑制剂等方法对其进行调控；并应用Flag-His串联亲和纯化方法获取USP18、ISG20靶蛋白，进一步研究该关键基因及蛋白的功能和在α干扰素抗HBV机制中的作用。本项目为国际合作研究，涉及功能基因组学与疾病发生发展机理等重大国际前沿，并试图从分子及细胞水平为探索慢乙肝防治的新思路作一些有益的尝试。

慢性乙型肝炎是我国的常见病和多发病，乙型肝炎慢性感染容易发生肝硬化、肝癌等不良后果，严重威胁我国及世界人民群众的身体健康。抗乙肝病毒药物中，α干扰素是抗HBV的首选药物之一。尽管如此，其昂贵的价格及效果欠佳等不利因素限制了其在临床上的广泛应用。因此，积极探索α干扰素抗HBV机制、提高其治疗有效率是临床亟待解决的难题。我们前期在宿主的基因水平筛选出了在α干扰素不同疗效组显著差异表达的两个关键基因USP18和ISG20，发现二者与α干扰素抗HBV治疗效果密切相关。研究表明USP18、CEB1等干扰素刺激基因在丙型肝炎患者α干扰素治疗前高表达常预示其对α干扰素治疗无应答。ISG20可以由I型、II型干扰素诱导产生，其抑制病毒复制主要依赖其核酸外切酶活性。ISG20蛋白产物可能具有抗HBV的效应，在HBV急性感染的黑猩猩中发现ISG20蛋白表达量明显增高。因此，我们推测ISG20可能不仅仅参与了α干扰素抗HBV途径，并且极有可能独立预测α干扰素对慢性乙肝的治疗效应。主要研究内容包括构建载体对USP18和ISG20在HepG2.2.15细胞内的表达进行调控，研究在靶基因表达变化的情况下，对α干扰素抗HBV活性的影响；给予不同浓度干扰素刺激，观察其参与抑制α干扰素抗HBV活性的机制及其在不同时间点参与抑制α干扰素抗HBV活性的机制及其在细胞内分布的变化。我们成功地构建了重组USP18 RNAi慢病毒载体并筛选出最有效地抑制USP18表达的重组慢病毒载体，转染HepG2.2.15细胞后，随着时间的推移和浓度的变化，对HBV的复制抑制作用越来越显著。而沉默组的ISG15、MxA、IFIT1蛋白表达增加。加入不同浓度的α干扰素以及处理不同时间后检测JAK/STAT信号通路相关因子的变化情况，证明了USP18和ISG20调控α干扰素的疗效与经典的JAK/STAT信号通路相关。通过调控USP18和ISG20的表达能增强α干扰素的抗病毒作用，为进一步探讨慢乙肝干扰素治疗提供了实验基础和理论依据，也为慢乙肝患者的治疗带来了新的希望。