【作者】 程玲；

【导师】 毛青；

【作者基本信息】 第三军医大学 ， 内科学， 2011， 硕士

【摘要】 丙型肝炎病毒(hepatitis C virus,HCV)感染是导致慢性肝炎、肝硬化甚至肝细胞癌(hepatocellular carcinoma,HCC)的主要原因之一。目前尚缺乏有效的疫苗,标准治疗方案是采用干扰素(interferon,IFN)联合利巴韦林(ribavirin,RBV),但部分患者疗效不理想。影响治疗效果的因素众多,包括宿主及病毒因素等。近年研究发现宿主基因多态性与HCV感染转归相关,相继报道了人类白细胞抗原(human leucocyte antigen,HLA)、IFN刺激基因和IL28B等。其中IL28B基因多态性对抗病毒治疗应答和病毒自然清除具有较好的预测价值。然而,在携带有危险等位基因的纯合子型人群中,仍有1/3的人获得了持续病毒学应答(sustained virological response,SVR),所以单纯考虑宿主因素并不能解释丙型肝炎抗病毒治疗的疗效,病毒对IFN治疗的影响也同样值得注意。HCV编码的多种病毒蛋白可通过影响细胞内众多信号传导级联反应而改变正常的应答途径导致IFN抵抗。相关机制研究较多,包括E2的糖基化结构抑制干扰素介导的蛋白激酶R(protein kinase R,PKR)通路以保护HCV不被清除;HCV NS3/4A影响干扰素调节因子3(interferon regulatory factor 3,IRF-3)信号传导,导致内源性免疫逃逸及外源性干扰素治疗抵抗;HCV NS5A蛋白C端2209-2248位氨基酸残基形成的干扰素敏感决定区(interferon sensitivity determining region,ISDR),与PKR结合后抑制PKR生物活性,但以上研究目前尚存在较大的争议。近年来研究认为HCV核心蛋白可作用于信号传导及转录激活因子(signal transducer and activator of transcription,STAT)家族,通过抑制JAK/STAT信号转导通路进而减少干扰素刺激基因(interferon stimulate gene,ISG)的转录;与核转录因子作用影响固有免疫应答因子,最终降低ISG表达。这些成果为深入研究HCV核心蛋白干扰素抵抗机制提供了大量的基础,使HCV核心蛋白成为研究HCV致病机制和防治的热点。本研究运用生物信息学工具预测HCV核心蛋白与IFN疗效相关的结构和功能,并通过临床观察研究验证,探讨HCV核心蛋白与IFN疗效相关的特征结构和功能区域中氨基酸变异及与IFN疗效的关系。实验方法和结果如下:一、HCV核心蛋白结构及功能的生物信息学预测利用生物信息学工具对HCV各基因型核心蛋白进行氨基酸序列分析及二、三级结构预测,并预测其可能存在的功能模序,综合分析比较HCV核心蛋白中存在的与IFN治疗敏感性相关的结构区域并分析型间差异。结果显示:1、HCV核心蛋白二级结构无规卷曲比例高而不易形成稳定的结构,2-45氨基酸可能存在螺旋-环-螺旋(helix-loop-helix)的三级结构。2、HCV核心蛋白16-36氨基酸可能与人解旋酶的DEAD box结构(DDX3)结合,2-23氨基酸可能与STAT1结合,可能与IFN抵抗有关。3、HCV核心蛋白中可能存在磷酸化等多种修饰位点及与SH2、SH3结构域结合的区域,可能调节不同信号转导通路影响IFN作用。二、HCV核心蛋白变异与干扰素疗效相关性的实验研究收集115例接受IFN联合RBV治疗的丙型肝炎患者并进行长期随访观察。治疗前进行HCV基因分型,并对患者血清HCV RNA进行动态监测,其中52例获得持续病毒学应答(SVR)和26例未获得持续病毒学应答(non-sustained virological response,N-SVR)的患者能成功扩增HCV核心(Core)区全长序列,对这部分患者Core区氨基酸序列进行比对分析,验证生物信息学预测中与IFN治疗敏感性相关的结构区域及各型间差异的结果,并进行氨基酸变异统计分析。1、HCV Core区结构功能预测结果与第一部分相符。各基因型间氨基酸变异主要集中在1-40和70-110氨基酸区域。2、aa4或aa91变异在SVR和N-SVR组的比较中有统计学意义,且Cys91变异的持续病毒学应答率有统计学意义。全文结论:1、HCV核心蛋白为二级、三级结构构象不稳定但可塑性好的蛋白,可能存在与DDX3和STAT等结合的区域及磷酸化、酰胺化等多种修饰位点和两性蛋白SH3结构域作用的区域。这些结构特点和功能区域均有可能影响IFN治疗效果,可能是核心蛋白抵抗IFN治疗的分子基础。2、HCV核心蛋白与IFN疗效相关的结构和功能区域中存在有统计学差异的氨基酸变异,推测这些氨基酸变异可能作为IFN治疗反应性的预测因子。更多还原

【Abstract】 Hepatitis C virus (HCV) usually causes chronic infection that can result in chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC), and there is no effective vaccine. The standard care for chronic hepatitis C (CHC) patients is combination therapy using interferon (IFN) and ribavirin (RBV). But the treatment outcome among patients is still unsatisfactory becauce of HCV’s resistance to interferon. In recent researchs found that host genetic variation was associated with reponse to IFN in HCV infection, such as human leucocyte antigen (HLA), interferon stimulate gene (ISG) and interleukin 28B (IL28B). The IL28B single-nucleotide polymorphisms (SNPs) were associated with the host immune response against HCV, contributed to clearance and IFN sensitivity. However, about 30 percent patients with danger allele could still receive sustained virological response (SVR), we should concert more factors that related to IFN sensitivity.HCV proteins played an important role in interferon resistance. E2 suppressed the protein kinase R (PKR), NS3/4A influenced interferon regulatory factor 3(IRF-3) to resist IFN, and interferon sensitivity determining region (ISDR) in NS5A may combined with PKR to suppresse activity, but there were some controversies. Recent researchs found that HCV core protein affected signal transducer and activator of transcription (STAT) and nuclear transcription factors. Resistance to IFN of HCV core protein becomes a research hotspot about HCV pathogenic mechanism and control these years. The aims of the study were to use different bioinformatics softwares to analyze and predict the structure and function correlated to interferon sensitivity of HCV core protein in different genotypes, then verified through the clinical observation research. Discussing the relationship between amino acid (aa) substitutions in the HCV core region and IFN antiviral sensitivity. Mean methods and results are as follows:1. Bioinformatics analysis of the structure and function of HCV core proteinUsed different bioinformatics softwares respectively to comparatively analyze and predict the amino acid sequences, secondary structure, tertiary structure, modification site and main functional motifs of HCV core protein. Comprehensively analyzed amino acid in HCV core region related to IFN antiviral sensitivity and differences in genotypes.1.1 HCV core protein secondary structure was largely unstructured and highly disordered, and tertiary structure at 2-45 amino acids existed helix-loop-helix.1.2 16-36 amino acids combined with DEAD-Box RNA Helicase (DDX3) and 2-23 amino acids combined with signal transducer and activator of transcription 1 (STAT1) would relate to IFN resistance.1.3 HCV core protein would presence some modification sites like phosphorylation, amidation, and areas combined with SH2, SH3 structure that could regulate signal transduction pathways to influence IFN response.2. Rresearch on the correlation between hepatitis C virus core protein mutation and interferon responseCollected 115 patients with chronic hepatitis C who received the IFN plus RBV therapy and genotyping, performed a long time follow-up with dynamic detection of their serum HCV RNA. Core region of 78 patients were successfully amplified, including 52 SVR and 26 N-SVR. Translated the core nucleotide into amino acid sequences, and analyzed the amino acid sequences to verify bioinformatics forecasting before.2.1 Prediction of structural and function of core protein from patients verified the bioinformatics forecasting before. And amino acid substitutions were mainly concentrated in 1- 40aa and 70 - 110 amino acids.2.2 There was statistically difference in aa4 or aa91 substitution between SVR group and N-SVR group, and Cys91 substitution had statistical significance in sustained virological response rate.Conclusions1. Secondary and tertiary structure of HCV Core protein was unstructure and disorder. Core protein would combine with DDX3 and STAT1 to influence IFN therapy, presence some modification sites and areas combined with SH2, SH3 structure to regulate signal transduction pathways to influence IFN response.2. Amino acid substitutions of HCV core protein related to IFN response would be an important predictor for IFN sensitivity.更多还原