【作者】 胡芸文；

【导师】 袁正宏； 张继明；

【作者基本信息】 复旦大学 ， 病原生物学， 2005， 博士

【摘要】 慢性丙型肝炎患者外周血单个核细胞干扰素及相关基因表达与干扰素疗效关系的研究丙型肝炎病毒(Hepatitis C Virus，HCV)感染引起的丙型肝炎仍是严重危害人民健康的重要病毒性疾病。丙型肝炎的主要危害性在于患者易转变为慢性肝炎，并可进一步发展为肝癌(HCC)而死亡。目前Ⅰ型干扰素(下简称干扰素)已被临床广泛用于慢性丙型肝炎的治疗，但尚存在治疗效果有限等缺点。虽然国内外对HCV基因结构、功能、致病性及机体免疫应答等已进行了大量研究，但迄今仍不十分清楚HCV与干扰素在机体和细胞内如何相互作用以影响机体对干扰素的应答。本室曾对丙型肝炎病毒的有关基因序列进行研究，结果未发现相关氨基酸序列与中国丙型肝炎病人的干扰素疗效有关。因此，有必要进一步探讨机体因素与干扰素疗效的相关性。HCV感染机体后可诱生机体产生内源性干扰素，同时又可抵抗干扰素的作用。当使用外源性干扰素对病人进行治疗时，机体对干扰素的应答实际上是病毒，内源性干扰素系统与外源性干扰素在体内相互作用、平衡的结果。理论推测，HCV可能通过基因复制和蛋白表达对干扰素通路上的关键环节进行干预，使内源性干扰素产生不足、信号通路受阻或干扰素的作用被拮抗，从而导致对干扰素的治疗不敏感；而由于宿主遗传背景因素差异引起的丙肝病毒感染者体内干扰素产生不足、干扰素诱导的信号转导通路缺陷或干扰素效应蛋白表达水平低下可能是其另一重要原因。为此，本研究以34例慢性丙型肝炎(chronic hepatitis C，CHC)病人为研究对象，在接受干扰素联合利巴韦林治疗前、治疗1个月、3个月、6个月、12个月和停药6个月时分别收集外周血标本，分离血清、血浆和外周血单个核细胞(PBMCs)后冻存于-80℃一并检测。根据疗程结束时和停药6个月时血清HCV RNA的检测结果，6例病人对干扰素非持续应答(包括无应答和复发的病人，简称NR)，28例病人则对干扰素持续应答(简称SR)。在此基础上采用实时定量聚合酶链式反应(Real-time PCR)方法分别对不同干扰素疗效病人PBMCs中Ⅰ型干扰素合成、信号转导以及抗病毒效应相关的基因的表达水平进行研究，并在丙肝病毒亚基因组复制细胞系统中验证有关结果；同时用基因芯片检测不同干扰素疗效病人PBMCs中全基因表达水平，以搜寻与疗效相关的基因表达特征(signature)或模式(pattern)。本研究首先探讨Ⅰ型干扰素及其合成相关基因的表达与HCV感染及干扰素疗效的关系。先检测了HCV慢性感染情况下PBMCs中IFN-α/β及其合成调节因子(IRF-1，IRF-3，IRF-5和IRF-7)的表达。结果发现，IFN-α和IFN-β的表达水平在病人和正常人(donor)之间没有显著的差异(P值分别为0.927和0.937)，而IRF家族成员的表达各不相同，其中IRF-3表达不受慢性感染的影响(P=0.980)，IRF-7表达在病人与正常人的差异未达到显著水平(P=0.071)，但在病人中IRF-1表达水平明显增强(P＜0.001)，IRF-5表达水平却显著低于正常人(P=0.002)。基因表达相关性分析显示，IRF-1的表达与IFN-α/β的表达无相关性；IRF-7的表达与IFN-α的表达显著相关(相关系数0.453；P值0.007)；IRF-5表达与IFN-α和IFN-β的表达则高度相关(相关系数0.686，0.621；P值＜0.001，0.001)。进一步分析Ⅰ型干扰素及其合成相关基因的表达与干扰素疗效的关系，发现上述相关基因在治疗前的表达水平与干扰素疗效无相关性，不能用于干扰素疗效的预测。为了确认HCV持续感染对IRF和Ⅰ型干扰素的影响，进一步在无HCV复制的Huh7细胞和HCV持续复制的Replicon细胞中研究IRF-7和IFN-α的表达水平。结果发现，在NDV攻击下，Replicon细胞的IRF-7 promoter活性、内源性IRF-7和IFN-α表达水平均较Huh7细胞低(IRF-7 promoter活性上调倍数：2.54 vs 4.20，P＜0.001；内源性IRF-7mRNA表达水平：0.137 vs 1.671，P＜0.001；内源性IFN-αmRNA表达水平：0.246 vs 2.207，P＜0.001)。但当Replicon细胞中的HCV复制被ribavirin和IFN-α降低或基本抑制后，IRF-7 promoter活性和内源性IRF-7、IFN-α表达水平均有不同程度的恢复。上述研究结果提示，在HCV慢性感染情况下，宿主Ⅰ型干扰素表达并无明显激活，部分干扰素合成调节因子的表达水平低下，可能为HCV易形成慢性感染的原因之一。此外，Ⅰ型干扰素及其合成相关基因在治疗前的表达水平与干扰素疗效无相关性，不能用于干扰素疗效的预测。干扰素诱导细胞内抗病毒的效应有赖于细胞内干扰素信号通路的效率，为此本研究进一步探讨了HCV慢性感染对病人PBMCs中干扰素信号通路正负调节基因表达的影响，并分析正负调节基因的平衡与干扰素疗效的关系。结果发现，HCV慢性感染者与正常人相比，JAK-STAT信号通路的正向调节因子STAT1和P48的表达和负向调节因子SOCS1和SOCS3的表达均显著增强(STAT1：CHC vs Donors 96.5 vs 69，P=0.039；P48：CHC vs Donors 121.5 vs 66.9，P=0.013；SOCS1：CHC vs Donors 5.96 vs 2.13，P=0.005；SOCS3：CHC vs Donors，78.3 vs 18.9，P＜0.001)；干扰素治疗前的SOCS1和SOCS5表达水平与HCV的复制水平呈负相关(R：-0.427和-0.385；P：0.013和0.027)。鉴于SOCS1和SOCS5主要表达于Th1细胞中，以上结果提示HCV复制可能会抑制Th1免疫的形成。进一步分析JAK-STAT信号通路正负调节因子的基因表达水平与干扰素疗效的相关性，发现治疗前负调因子SOCS3的高表达与干扰素的疗效不佳有关(NR vs SR，203.0 vs 64.1，P=0.007)，推测SOCS3的高表达削弱了细胞对外源性干扰素的反应能力。鉴于SOCS3主要在Th2细胞中表达，非持续应答病人治疗前的PBMCs中SOCS3的高水平表达也提示在Th2类免疫状态下机体对干扰素反应低下。利用流式细胞仪分析应答病人在干扰素治疗前后PBMCs中γ-干扰素和IL-4分泌细胞的水平，结果与SOCS的水平相一致：在干扰素治疗过程中，随着血清中HCV RNA的下降，SOCS1和SOCS5表达上调而SOCS3表达下调，Th1和Th2细胞百分含量也分别呈现增加和减少(Th1：治疗前vs治疗3个月，4.28+2.4％vs 7.63±3.5％P＜0.001；Th2：治疗前vs治疗3个月，0.66±0.52％vs 0.37±0.53％，P=0.013)，提示其可用于干扰素免疫调节效果的监测。进一步在体外肝细胞系中确认HCV蛋白表达和基因复制对肝细胞内SOCS1和SOCS3表达的影响，发现HCV编码的Core蛋白可诱导Huh7细胞表达SOCS1和SOCS3，尤其以SOCS1表达最为显著，其它HCV的非结构蛋白则无此诱导能力。据于外源性干扰素治疗丙型肝炎的效果主要取决于能否诱导足够的抗病毒效应分子的产生，本研究进一步检测了抗病毒效应分子MxA，PKR和2’-5’OAS在干扰素治疗前及治疗过程中的表达，并分析其基因表达与疗效的关系。结果发现，病人PBMCs中MxA，PKR和2’5’OAS的表达在干扰素治疗前与对照相比已有明显增强(MxA：CHC vs Donors 141.4 vs 5.5，P＜0.001；PKR：CHC vs Donors 75.7 vs 12.3，P＜0.001；2’-5’OAS：CHC vs Donors 72.6 vs 4.18，P＜0.001)，干扰素治疗过程中其表达水平进一步升高，但其治疗前的基础表达水平和治疗中的诱导表达水平均与干扰素疗效无关，提示MxA，PKR和2’-5’OAS的基因表达不能用于疗效的预测。进一步用基因芯片(U-133A，Affymetrix公司)的方法检测了干扰素持续应答者和非持续应答者的PBMCs在体外用干扰素诱导后的全基因表达，发现不同疗效组的基因表达大部分相似，仅有16个基因的诱导表达倍数在不同疗效组中有显著性差异，包括已知的干扰素刺激基因(STAT1，GBP1，IFIT1，GIP3和IFIT2)，趋化因子基因(CXCL10和CCL8)，信号通路相关蛋白基因(STK3，RASGRP3，RIN2和LEPR)，代谢相关蛋白基因(APOBEC3A，MANEA和ZC3HAV1)和功能尚不明确的基因(REC8L1和C6orf62)。综上所述，本研究的结果表明，HCV慢性感染对PBMCs中Ⅰ型干扰素及其相关基因的表达产生影响。HCV慢性感染情况下，Ⅰ型干扰素及其合成关键因子的表达受到抑制，治疗前的表达水平与疗效无关；干扰素信号通路的正负调节因子的表达均被增强，并且负调因子SOCS3的表达水平与疗效相关；抗病毒效应分子MxA，PKR和2’-5’OAS的基础表达增强，基础表达和诱导表达水平均与疗效无关，同时基因组学研究发现PBMCs中16个基因在干扰素诱导下的表达变化与疗效相关。HCV慢性感染干预宿主细胞基因表达的有关机制有待进一步利用细胞系统进行深入研究；宿主基因表达与临床干扰素疗效的相关性研究及其在指导临床用药等方面的应用，需要在更大的病人样本中探索和实践。本课题的研究结果一方面能丰富人们对干扰素诱导抗病毒及HCV拮抗干扰素机制的认识，另一方面可指导临床预测疗效、筛选病人和制定治疗方案，最终实现个性化的干扰素治疗，达到提高丙型肝炎治愈率的目的。更多还原

【Abstract】 Hepatitis C virus (HCV) is estimated to infect more than 170 million people worldwide. HCV infection usually causes chronic hepatitis in approximately 80% of infected individuals with 20% progressing to cirrhosis and 1-2% developing hepatocellular carcinoma. Currently, Interferon-α (IFN-α) is one of the most efficient drugs for the treatment of chronic hepatitis C and the standard treatment for HCV infection is IFN-α in combination with ribavirin. Unfortunately, more than 60% of patients with chronic HCV infection will experience either no control of viral replication by therapy (nonresponders) or a relapse when therapy is stopped (relapsers). The molecular mechanisms underlying failure of IFN-α treatment are not well understood. As our previous research found no correlation between viral genomic sequence and outcome of IFN-α treatment, host factors associated with IFN therapy, were explored in this study.It has been proposed that HCV has the ability of interfering with type I interferon (IFN-α/β) production, blocking JAK-STAT signaling transduction, inhibiting expression or activity of ISGs (interferon -stimulated genes), which may support HCV replication and persistence as well as resistance to IFN-α therapy. Host gene expression is the outcome of host reaction to infection and viral interference with host, and may correlate with efficiency of IFN-α treatment. To identify signature or pattern of host gene expression which can distinguish different outcome of therapy, expression of type I interferon, interferon regulated factors (IRFs), positive and negative regulatory factors of IFN signaling, classical antiviral effectors and whole cellular genes were analyzed in peripheral blood mononuclear cells (PBMCs) from 34 chronic hepatitis C (CHC) patients before, during and after IFN/ribavirin treatment byreal-time quantitative RT-PCR and microarray. Among them, 6 of 34 were nonresponders (NR) and 28 of 34 were sustained responders (SR) according to serum HCV RNA test at the end of treatment and 6 months post-treatment. Gene expression was compared between these two groups.To identify the correlation between gene expression of type I interferon, IRFs and outcome of IFN therapy, gene expression level of IFN-α/β, IRF-1, IRF-3, IRF-5 and IRF-7 were quantified in CHC patients. Result showed that, IFN-α and IFN-β expression level in the patients and donors were not significantly different (P: 0.927 and 0.937). However, IRF family members were differentially expressed in the two patient groups. IRF-3 expression was not influenced by chronic infection (P=0.980) and IRF-7 expression was not significantly different between patients and donors(P=0.071); IRF-1 expression levels were obviously enhanced in patients(P<0.001) and IRF-5 expression level was remarkably lower than that of the donors (P=0.002). The relevance analysis of gene expression showed that, while IRF-1 expression and the IFN-α/β expression did not have relevance, IRF-7 expression and IFN-α expression were correlated (R= 0.453; P= 0.007) and IRF-5 expression correlated with expression of both IFN-α and IFNβ (R=0.686, 0.621; P <0.001, 0.001). This result may reflect the mechanism of low expression of host type I interferon, which might be associated with low expression of key factors that regulated interferon’s synthesis. However, further analysis did not reveal any correlation between gene expression of IFN-α/β, IRF-1, IRF-3, IRF-5 and IRF-7 before treatment and responses of host to interferon treatment. In order to confirm the influence of HCV persistent infection on IRF and type I interferon, HCV replicon cell (Huh7 cell line harboring subgenomic HCV replicons) was used. Result showed that, under NDV attack, IRF-7 promoter activity or the endogenous IRF-7 and IFN-α expression level of HCV replicon cells was lower than that of Huh7 cell (fold activity of IRF-7 promoter: 2.54 vs. 4.20, P<0.001; endogenous IRF-7mRNA expression level: 0.137 vs. 1.671, P<0.001; endogenous IFN-α mRNA expression level: 0.246 vs 2.207, P<0.001). On inhibition of HCV replication by ribavirin or IFN-α treatment, both IRF-7 promoter activity and expression of endogenous IRF-7and IFN-α were restored. This result may suggest a new mechanism of inhibition of type I interferon by HCV.As the interferon-induced intracellular antiviral effect depends largely on efficiency of signal transduction (JAK-STAT pathway), the effect of HCV chronicinfection on expression of positive and negative regulatory genes of JAK-STAT pathway in patients’ PBMCs was further analyzed. Results showed that, compared to healthy donors, the expressions of positive regulatory genes (STAT1 and P48) and negative regulatory genes (SOCS1 and SOCS3) were markedly enhanced in CHC patients (STAT1: CHC vs Donors 96.5 vs. 69, P=0.039; P48: CHC vs. Donors 121.5 vs. 66.9, P=0.013; SOCS1: CHC vs. Donors 5.96 vs. 2.13, P=0.005; SOCS3: CHC vs. Donors 78.3 vs. 18.9, P<0.001). Expression level of SOCS1 and SOCS5 before treatment was negatively correlated with serum HCV RNA level (R= -0.427 and -0.385; P= 0.013 and 0.027). As SOCS1 and the SOCS5 are mainly expressed in the Thl cell, the above result indicated that HCV replication might be able to suppress the Thl immunity. Further analysis of relevance between expression of these key factors of signaling pathway and outcome of IFN therapy showed high SOCS3 expression was predictive of no response to IFN therapy (NR vs. SR 203.0 vs. 64.1, P=0.007). It indicates that SOCS3 expression level may be used as marker for prediction of IFN-α therapy. Using flow cytometer to analyze the cytokine secreting cells, we found a switch-over of cytokine synthesis from Th2 to Th1 (IFN-γ for Th1, IL-4 for Th2) (Th1: 0 week vs. 12 weeks 4.28±2.4% vs. 7.63±3.5%, P<0.01; Th2: 0 week vs. 12 weeks 0.66±0.52% vs. 0.37±0.53%, P= 0.013) during IFN-α therapy, which was paralleled to up-regulation of SOCS1 and SOCS5 level and down-regulation of SOCS3 level. This result indicated that SOCS expression level in PBMC might be used to evaluate immune regulatory effect of IFN-α treatment. In vitro assay in cell line further showed that the expression of SOCS1 and SOCS3 in Huh7 cell line were induced by HCV Core protein, but not by other HCV non-structural proteins.As antiviral effect of interferon mainly depends on induction of antiviral effectors molecule, gene expression of classical antiviral effectors MxA, PKR and 2 ’ 5 ’ OAS before and during treatment and its correlation with outcome of therapy were further analyzed. Results showed that, compared with healthy donors, MxA, PKR and 2 ’ 5 ’ OAS expression in patients were obviously enhanced (MxA: CHC vs. Donors 141.4 vs. 5.5, P<0.001; PKR: CHC vs. Donors 75.7 vs. 12.3, P<0.001; 2’5’OAS: CHC vs. Donors 72.6 vs. 4.18, P<0.001) and were further elevated by IFN-α treatment. However, there was no correlation of both basal expression levels and fold induction during treatment of the above genes with outcome of IFN treatment, which suggest that MxA, PKR and 2’5’ OAS gene expression cannot be used as predictive marker for IFN therapy. DNA microarrays(Affymetrix Com, HG U-133A, containing 13000cellular genes and EST) were further used to analyze the global gene expression of PBMCs in NR and SR groups. Comparing the fold of induction of genes from these two groups, we found that 16 genes were differentially expressed between NR and SR. These genes included ISGs (STAT1, GBP1, IFIT1, GIP3 and IFIT2), chemokines (CXCL10 and CCL8), genes related with signaling pathway (STK3, RASGRP3, RIN2 and LEPR), metabolism-correlated gene (APOBEC3A, MANEA and ZC3HAV1) and several genes with uncertain function (REC8L1 and C6orf62).In summary, our research results indicated that HCV chronic infection has the influence on expression of type I interferon and its related genes in PBMCs. Under HCV chronic infection situation, while expression of type I interferon and its key regulatory factor was suppressed, the expression of the positive and negative regulatory factors of interferon signaling pathway were strengthened. Furthermore, while the expression of type I interferon, its key regulatory factor and MxA, PKR, 2’5’OAS before treatment have no relationship with outcome of IFN-α treatment, there was correlation of expression of negative factor SOCS3 with outcome of IFN-α treatment. In addition, 16 cellular genes in PBMCs were differentially expressed between NR and SR. Further study with HCV replicon cells is needed to understand the molecular mechanism underlying intervention of host cell gene expression by HCV chronic infection. The possible application of host gene expression pattern in guiding clinical interferon treatment such as predicting responses to IFN treatment need to be further investigated in a larger cohort of CHC patients. These findings, on the one hand, can enrich our knowledge about interaction between HCV and host defense system; on the other hand, may help to optimize clinical treatment and improve efficacy of IFN, and finally realize the individualized interferon treatment for HCV patients.更多还原