由乙型肝炎病毒(Hepatitis B virus，HBV)感染引起的慢性乙型肝炎是严重危害人类健康的疾病，据估计，全世界大约有三亿五千万乙肝患者，慢性乙肝能进一步发展为肝硬化、肝细胞性肝癌(HCC)而导致死亡。国内外对HBV基因结构、功能、致病性及与机体免疫应答的关系等已有了大量的研究，然而大多数研究主要聚焦于获得性免疫在HBV致病性和免疫应答中的作用，如发现HBV特异性T细胞的耗竭是HBV不易被免疫清除的主要原因之一。然而，对于HBV感染与天然免疫应答尤其是Toll样受体(Toll-like receptor，TLR)信号通路的相互作用，目前尚不明确。 为了研究HBV与TLR信号通路的相互作用，本课题首先利用实时荧光定量PCR技术检测了慢性乙型肝炎患者与正常人外周血单个核细胞(PBMCs)中TLR1-10基因表达水平的差异。结果发现，患者PBMCs中TLR1、TLR2、TLR4和TLR6基因的表达水平较正常人有2倍以上的下调。进一步通过流式细胞术检测了患者和正常人PBMCs表面TLR2和TLR4的表达，结果显示与正常人相比，患者PBMCs表面TLR2蛋白的表达有显著的下降，而TLR4的表达没有显著的差异。为了评价患者的HBsAg、HBeAg水平，HBV病毒载量，HBV基因型， ALT水平以及疾病持续时间等因素对TLR表达的影响，将TLR基因表达水平与上述指标进行了非条件多因素logistic回归分析。结果显示，HBV C型的患者PBMCs上TLR2的表达水平低于HBV B型患者，而其它的临床指标和TLR表达没有显著的相关性。 为研究患者和正常人的PBMCs中TLR2和TLR4的表达差异是否具有功能学上的意义，进一步利用TLR2和TLR4的配体(Pam3csk4和LPS)刺激PBMCs，微量样本多指标流式蛋白定量方法(CBA)检测其产生细胞因子IL-1β、IL-6、 IL-8、IL-10、IL-12和TNF-α的能力。结果发现，无论是TLR2配体还是TLR4 配体刺激，患者PBMCs分泌细胞因子如IL-6、IL-8、IL-12和TNF-α的能力均明显降低。在此基础上，对患者PBMCs受TLR2配体、TLR4配体诱导产生细胞因子的能力与患者外周血中的HBsAg、HBeAg水平，HBV病毒载量，ALT水平等进行了相关性分析。结果提示，患者PBMCs产生细胞因子的能力受损和患者外周血中高水平的HBsAg具有部分相关性，提示患者体内细胞因子表达的下降可能是由于HBsAg的大量存在所致。 在发现患者PBMCs受TLR2和TLR4配体刺激后产生细胞因子的能力受损，并与HBsAg可能相关的基础上，进一步对HBsAg是否能够抑制TLR2和4信号传导通路以及其分子机制作深入研究。以单核细胞THP-1经乙酸肉豆蔻佛波醇(PMA)诱导后的巨噬样细胞为模型，首先在体外验证了 HBsAg对这两条信号通路所诱导的细胞因子产生的影响。结果发现，无论是TLR2配体还是TLR4配体的刺激，HBsAg均能以剂量依赖的方式抑制IL-12 p40的产生，包括mRNA水平和蛋白水平，而对IL-10的产生没有显著的影响。HBsAg对TLR4信号通路的抑制作用与前期其他学者的报道相符合，对于这一现象的机制研究也已有一定进展。由于HBsAg对TLR2信号通路的影响，目前还未见到相关的报道，进一步将工作集中于TLR2信号通路。 为了更全面地了解HBsAg对TLR2配体诱导的细胞因子产生的影响，检测了HBsAg处理及未处理情况下，THP-1经PMA诱导分化成巨噬样细胞受TLR2配体刺激其各细胞因子IL-1β、IL-6、IL-8、IL-10、IL-12和TNF-α的产生水平。结果发现，HBsAg能够选择性地抑制TLR2配体诱导的IL-12的产生，而对IL-6、 IL-8和TNF-α的产生没有显著的影响。 为探讨HBsAg抑制IL-12产生的可能的分子机制，首先检测了HBsAg对TLR2的表达是否存在影响。流式结果显示，HBsAg能够显著下调巨噬样细胞表面TLR2的表达水平。通过Western blot以及激光共聚焦的方法进一步检测了HBsAg对与诱导IL-12产生相关的TLR2信号通路下游包括NF-κB和MAPK两条信号通路的影响。研究结果显示，HBsAg对TLR2配体诱导的NF-κB信号通路中包括IκB-α蛋白的磷酸化和降解没有显著的影响，对NF-κB亚基p65的入核亦没有显著的影响；对TLR2配体诱导的MAPK信号通路中p38、ERK等蛋白的磷酸化也没有显著的影响。然而，HBsAg能够抑制TLR2配体诱导的JNK的磷酸化，并能够进一步抑制JNK激酶下游转录因子c-Jun的磷酸化。同时应用JNK磷酸化的抑制剂SP600125，发现能够特异性地抑制JNK的磷酸化以及IL-12的分泌，而对p38和ERK的磷酸化没有影响，间接证明了 HBsAg可能通过影响TLR2配体诱导的JNK磷酸化，从而抑制IL-12的分泌。 综上所述，本研究通过对慢性乙型肝炎患者PBMCs的研究，发现TLR2与TLR4的功能性表达受到HBV感染的影响，并发现与患者血清HBsAg水平呈现负相关；进一步的体外实验研究发现HBsAg能够选择性地抑制TLR2配体诱导的IL-12的分泌，并且这种抑制作用可能是由于其特异性地抑制了下游MAPK信号通路中JNK的磷酸化所引起。上述研究结果有助于增进HBV感染与宿主天然免疫之间的相互作用的了解，并可为开发新一代的抗HBV制剂或药物提供实验和理论依据。 关键词：慢性乙型肝炎；Toll样受体：细胞因子；乙肝表面抗原；MAPK信号传导通路 中图分类号：R373.2+1

 Chronic hepatitis B caused by hepatitis B virus (HBV) infection is one of the most prevalent liver diseases in the world, affecting more than 350 million people. Chronic hepatitis B (CHB) usually leads to fulminant hepatic failure, liver cirrhosis, and primary hepatocellular carcinoma, which results in more than 1200,000 deaths per year. Remarkable strides have been made in the understanding of the genome, function, pathogenesis and host immune response of hepatitis B virus. Most studies have underlined the importance of adaptive immunity in HBV infections. For example, patients with chronic hepatitis B tend to have late, transient or narrowly focused T-cell response, which indicated that HBV maintains viral persistence by clonal exhaustion of HBV-specific T cell. However, for the role of innate immune response, especially TLRs, against HBV, there are only a few studies and the results are not consistent. In order to study the interplay between HBV and the TLR signal pathway, we firstly measured the TLR1-10 specific transcripts in PBMCs from chronic HBV infected patients and healthy donors by quantitative real-time PCR. Results showed that PBMCs from patients with chronic HBV infection expressed significantly lower levels of TLR1, TLR2, TLR4 and TLR6 mRNA transcripts compared with those from healthy donors. Besides, using flow cytometric analysis, we found that the cell surface expression of TLR2 on PBMCs was significantly decreased in chronic HBV infected patients compared to the healthy donors, while the cell surface expression of TLR4 showed no statistically significant difference. Unconditional multivariate logistic analysis were further performed to evaluate the effect of independent factors such as plasma levels of HBsAg, HBeAg, HBV viral load, HBV genotype, alanine aminotransferase (ALT) level and the duration of chronic infection on the expression of TLR1, TLR2, TLR4 and TLR6. Result showed that HBV genotype was the only significant factor to TLR2 expression. And patients with genotype C expressed lower level of TLR2 compared with those with genotype B. Other clinical variables were not significantly related to TLR expression. To evaluate whether PBMCs from chronic HBV infected patients are defective in cytokine production, we investigated the ability of PBMCs from CHB patients and healthy donors to produce cytokines in response to TLR2 and TLR4 ligands (Pam3csk4 and LPS). The amounts of inflammatory cytokines (e.g. IL-1β, IL-6, IL-8, IL-10, IL-12 and TNF-α) in the supematants of PBMCs were quantitated using cytometric bead array. After challenged with Pam3csk4 and LPS, significantly lower level of IL-6, IL-8, IL-12 and TNF-α production from PBMCs of CHB patients were observed, compared with those from healthy donors. But, there was no significant difference in IL-1β and IL-10 production between these two groups. In addition, we evaluated whether impaired cytokine production was correlated with patients' clinical parameters, including the plasma levels of HBsAg, HBeAg, viral count and ALT. Results showed that the Pam3csk4 and LPS induced production of cytokine was partially inversely correlated with the level of HBsAg in CHB patients, which indicated that the impaired cytokine production might be due to the high level of HBsAg. Based on the above results, further studies were carried out to study the role of HBsAg in modulation of TLR signal pathway. Phorbol 12-myrisate 13-acetate (PMA) induced THP-1 cell was used in this study as a model for human macrophage. We firstly examined cytokine production in PMA induced macrophages derived from human monocyte THP-1 cells after stimulated by Pam3csk4 and LPS. Results showed that HBsAg could inhibit Pam3csk4 and LPS induced IL-12 p40 production in a dose-dependent manner. In addition, suppression of IL-12 production occurred at the transcriptional level by reducing IL-12 p40 mRNA level. However, HBsAg did not have significant effect on production of IL-10. As it has been reported that HBsAg could inhibit the release of LPS-induced cytokines in human monocytes, we focused on the TLR2 signal pathway and the mechanism by which HBsAg affect monocyte function. To determine whether HBsAg could affect the production of other cytokines, we examined the effect of HBsAg on secretion of IL-1β, IL-6, IL-8, IL-10, IL-12 and TNF-α induced by TLR2 ligand in PMA induced THP-1 cells. Results showed that HBsAg selectively inhibited IL-12 production in macrophage in response to Pam3csk4 stimulation. To further investigate the mechanism by which HBsAg selectively inhibited IL-12 production, firstly, the TLR2 expression affected by HBsAg was analyzed by FACS. Results showed that HBsAg could down-regulated TLR2 expression in THP-1 induced macrophages. Furthermore, the NF-κB and MAPK signal pathways, which are important for IL-12 production, were analyzed by Western blot and immunofluorsescence staining. We found that HBsAg did not interfere with the phosphorylation and degradation of IκB-α, and the number of cells positive for nuclear NF-κB p65 was not significantly different between the HBsAg-treated cells and the control cells. We also found that HBsAg did not interfere with the phosphorylation of p38 and ERK-1/2. Interestingly, the Pam3csk4-induced phosphorylation of JNK-1/2 and c-Jun, a transcription factor of JNK pathway, were reduced in the presence of HBsAg. In addition, the role of JNK was confirmed by using specific inhibitor of JNK activation, SP600125. SP600125 specifically inhibited Pam3csk4-induced JNK activation and IL-12 production, while it did not inhibit the phosphorylation of either p38 or ERK-1/2 MAPKs. Taking together, these results indicated that HBsAg may inhibit IL-12 production in Pam3csk4-induced human monocytic cells by down-regulating the activation of JNK MAPK pathway. In conclusion, our ex vivo studies demonstrated that functional expression of TLR2 and TLR4 were down-regulated by HBV infection, which correlated with the increased level of HBsAg. Further in vitro studies found that HBsAg selectively inhibited IL-12 production in macrophage in response to Pam3csk4 stimulation, which might be caused by interfering with the JNK MAPK pathway. These findings may facilitate the understanding of mechanism of HBV chronic infection, especially the role of innate immune response in HBV infection and may help to design novel therapeutic targets for chronic hepatitis B. Keywords: Chronic hepatitis B virus infection; Toll-like receptor; cytokine; hepatitis B virus surface antigen; MAPK signal pathway;