乙型肝炎病毒干预后小鼠肝脏树突状细胞干扰素调节因子3表达变化的研究
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摘要
目的：
通过观察乙型肝炎病毒干预后小鼠肝脏树突状细胞干扰素调节因子3表达的变化,探讨HBV持续感染分子免疫机制。
方法：
采用细胞滤网过滤、Percoll密度梯度离心和免疫磁珠分选法分离肝脏树突状细胞,并用粒细胞—巨噬细胞集落刺激因子和白介素4诱导培养。将树突状细胞分为两组：一组与HBV共培养,另一组加入等量的细胞培养液作为正常对照组。24h后,两组均加入Poly I:C刺激,收集不同时间点细胞和上清,留细胞培养液作为空白对照,用western blot检测IRF3表达,用ELISA法检测上清中IFN-β浓度。
结果：
1.DC与HBV共培养前,IRF3表达弱。用poly I:C刺激DC后,IRF3表达明显增加,呈时相性,在2-6h达到高峰。DC与HBV共培养后,再用poly I:C刺激,IRF3表达的时相性不明显,未见明显升高。DC表达IRF3的能力与病毒载量有关,高病毒载量能明显抑制IRF3表达,病毒载量低于106 copies／ml时差异不明显。
2.0h时HBV组培养液上清IFN-β浓度(12.38±3.71pg／ml)与正常对照组(10.83±4.11 pg／ml)相比,差异无统计学意义(t=0.8398,P>0.05)。Poly工：C刺激后6h,HBV组培养液上清IFN-β浓度(88.67±9.01 pg／ml)与正常对照组(137.68±12.28 pg／ml)相比,差异有统计学意义(t=9.653,P<0.01)。Poly I:C刺激后24h,HBV组培养液上清IFN-β浓度(69.89±5.80 pg／ml)与正常对照组(72.25±8.61 pg／ml)相比,差异无统计学意义(t=0.6820,P>0.05)。
结论：
1.用poly I:C刺激正常DC后,IRF3表达增加,呈时相性。
2.HBV干预后,用poly I:C刺激DC后,IRF3表达增加不明显。高载量HBV能明显抑制肝脏树突状细胞IRF3的表达。
3.HBV干预后,肝脏树突状细胞IRF3下游IFN-β表达降低。
Objectives:
To investigate the molecule immunology mechanism of HBV persistent infection, we observed the expression of IRF3 in murine liver dendritic cells, which were intervened by HBV virions.
Methods:
The murine liver dendritic cells were isolated via anti-CD11c microbeads, and were incubated with GM-CSF and IL-4 to induce the DCs generation and proliferation. DCs were divided into two groups. One group was cultured with HBV virions in 24h; the other group was cultured without HBV as a normal control group. Then, harvesting the cells and supernatants were detected the expressions of p-IRF3 by western blot and the concentration of IFN-βby ELIS A, after stimulated by poly I:C.
Results:
1. The expression of IRF3 was too weak to be detected by western blot, before dendritic cells were co-incubation with HBV. The expression of IRF3 was increased after normal dendritic cells were stimulated by poly I:C. The highest expression time-point was between 2h to 6h. The expression of IRF3 was not significantly increased after dendritic cells were incubated with HBV. The ability of expression of IRF3 of DCs was correlated with HBV viral load. High HBV viral load can significantly inhibit expression of IRF3. HBV did not inhibit IRF3 expression significantly, if HBV viral load was lower than 106 copies/ml.
2. IFN-βconcentration was 12.38±3.71pg/ml at Oh,88.67±9.01 pg/ml at 6h, 69.89±5.80 pg/ml at 24h in supernatants of HBV group, and was 10.83±4.11 pg/ml at 0h,137.68±12.28 pg/ml at 6h,72.25±8.61 pg/ml at 24h in supernatants of normal control group, respectively. There was no statistically significant difference at 0h(t=0.8398, P>0.05) and at 24h(t=0.6820, P>0.05) between two groups. But, there was statistically significant difference at 6h(t=9.653, P<0.01) between two groups.
Conclusion
1. The expression of IRF3 was increased after normal dendritic cells were stimulated by poly I:C and had a relationship with different stimulated time-point.
2. After co-incubation with HBV, the expression of IRF3 in liver DCs was not increased significantly, compared with normal control. High HBV viral load can inhibit the expression of IRF3 significantly.
3. The secretions of IFN-β, downstream of IRF3, were decreased in murine liver dendritic cells after intervened by HBV virions.
通过观察乙型肝炎病毒干预后小鼠肝脏树突状细胞干扰素调节因子3表达的变化,探讨HBV持续感染分子免疫机制。
方法：
采用细胞滤网过滤、Percoll密度梯度离心和免疫磁珠分选法分离肝脏树突状细胞,并用粒细胞—巨噬细胞集落刺激因子和白介素4诱导培养。将树突状细胞分为两组：一组与HBV共培养,另一组加入等量的细胞培养液作为正常对照组。24h后,两组均加入Poly I:C刺激,收集不同时间点细胞和上清,留细胞培养液作为空白对照,用western blot检测IRF3表达,用ELISA法检测上清中IFN-β浓度。
结果：
1.DC与HBV共培养前,IRF3表达弱。用poly I:C刺激DC后,IRF3表达明显增加,呈时相性,在2-6h达到高峰。DC与HBV共培养后,再用poly I:C刺激,IRF3表达的时相性不明显,未见明显升高。DC表达IRF3的能力与病毒载量有关,高病毒载量能明显抑制IRF3表达,病毒载量低于106 copies／ml时差异不明显。
2.0h时HBV组培养液上清IFN-β浓度(12.38±3.71pg／ml)与正常对照组(10.83±4.11 pg／ml)相比,差异无统计学意义(t=0.8398,P>0.05)。Poly工：C刺激后6h,HBV组培养液上清IFN-β浓度(88.67±9.01 pg／ml)与正常对照组(137.68±12.28 pg／ml)相比,差异有统计学意义(t=9.653,P<0.01)。Poly I:C刺激后24h,HBV组培养液上清IFN-β浓度(69.89±5.80 pg／ml)与正常对照组(72.25±8.61 pg／ml)相比,差异无统计学意义(t=0.6820,P>0.05)。
结论：
1.用poly I:C刺激正常DC后,IRF3表达增加,呈时相性。
2.HBV干预后,用poly I:C刺激DC后,IRF3表达增加不明显。高载量HBV能明显抑制肝脏树突状细胞IRF3的表达。
3.HBV干预后,肝脏树突状细胞IRF3下游IFN-β表达降低。
Objectives:
To investigate the molecule immunology mechanism of HBV persistent infection, we observed the expression of IRF3 in murine liver dendritic cells, which were intervened by HBV virions.
Methods:
The murine liver dendritic cells were isolated via anti-CD11c microbeads, and were incubated with GM-CSF and IL-4 to induce the DCs generation and proliferation. DCs were divided into two groups. One group was cultured with HBV virions in 24h; the other group was cultured without HBV as a normal control group. Then, harvesting the cells and supernatants were detected the expressions of p-IRF3 by western blot and the concentration of IFN-βby ELIS A, after stimulated by poly I:C.
Results:
1. The expression of IRF3 was too weak to be detected by western blot, before dendritic cells were co-incubation with HBV. The expression of IRF3 was increased after normal dendritic cells were stimulated by poly I:C. The highest expression time-point was between 2h to 6h. The expression of IRF3 was not significantly increased after dendritic cells were incubated with HBV. The ability of expression of IRF3 of DCs was correlated with HBV viral load. High HBV viral load can significantly inhibit expression of IRF3. HBV did not inhibit IRF3 expression significantly, if HBV viral load was lower than 106 copies/ml.
2. IFN-βconcentration was 12.38±3.71pg/ml at Oh,88.67±9.01 pg/ml at 6h, 69.89±5.80 pg/ml at 24h in supernatants of HBV group, and was 10.83±4.11 pg/ml at 0h,137.68±12.28 pg/ml at 6h,72.25±8.61 pg/ml at 24h in supernatants of normal control group, respectively. There was no statistically significant difference at 0h(t=0.8398, P>0.05) and at 24h(t=0.6820, P>0.05) between two groups. But, there was statistically significant difference at 6h(t=9.653, P<0.01) between two groups.
Conclusion
1. The expression of IRF3 was increased after normal dendritic cells were stimulated by poly I:C and had a relationship with different stimulated time-point.
2. After co-incubation with HBV, the expression of IRF3 in liver DCs was not increased significantly, compared with normal control. High HBV viral load can inhibit the expression of IRF3 significantly.
3. The secretions of IFN-β, downstream of IRF3, were decreased in murine liver dendritic cells after intervened by HBV virions.
引文
1 Lok AS, McMahon BJ. Chronic Hepatitis B:Update 2009. Hepatology 2009;50(3):661-662.
2 Liu FM, Zhuang H. Management of hepatitisB in China. Chin Med J 2009; 122: 1-2.
3 Tsai SL, Huang SN. T cell mechanisms in the immunopathogenesis of viral hepatitis B and C. J Gastroenterol Hepatol 1997;12(9-10):S227-235.
4 Hugues S, Boissonnas A, Amigorena S, et al. The dynamics of dendritic cell-T cell interactions in priming and tolerance. Curr Opin Immunol 2006; 18(4):491-495.
5 Ito T, Amakawa R, Fukuhara S. Roles of toll-like receptors in natural interferon-producing cells as sensors in immune surveillance. Hum Immunol 2002;63(12):1120-1125.
6 Beckebaum S, Cicinnati VR, Zhang X, et al. Hepatitis B virus-induced defect of monocyte-derived dendritic cells leads to impaired T helper type 1 response in vitro: mechanisms for viral immune escape. Immunology 2003;109(4):487-495.
7陈明泉,施光峰,卢清等.不同病毒载量的慢性乙型肝炎患者外周血树突状细胞的表型和功能.中华肝脏病杂志2007；15(1)：19-23.
8李谦,陈明泉,李宁等.聚肌胞刺激后慢性乙型肝炎患者树突状细胞内Toll样受体3的表达变化.中华传染病杂志2009；27(12)：733-737.
9张玉杰,施光峰,李谦等.慢性乙型肝炎患者树突状细胞Toll样受体3触发后Ⅰ型干扰素的表达.中华传染病杂志2009；27(6)：343-347.
10安宝燕,谢青,王晖等.干扰素调节因子3在慢性乙型肝炎患者外周血树突状细胞中的表达及意义.世界华人消化杂志2008；16(17)：1873-1879.
11 Seth RB, Sun L, Ea CK, et al. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NFkappaB and IRF 3. Cell 2005;122:669-682.
12 Kawai T, Takahashi K, Sato S, et al. IPS-1, an adaptor triggering RIG-I-and Mda5-mediated type I interferon induction. Nat Immunol 2005;6:981-988.
13 Meylan E, Curran J, Hofmann K, et al. Cardif is an adaptor protein in the RIG-Ⅰ antiviral pathway and is targeted by hepatitis C virus. Nature 2005;437:1167-1172.
14 Hart DN, Fabre JW. Demonstration and characterization of la-positive dendritic cells in the interstitial connective tissues of rat heart and other tissues, but not brain. J Exp Med 1981;154(2):347-361.
15 Prickett TC, McKenzie JL, Hart DN. Characterization of interstitial dendritic cells in human liver. Transplantation 1988;46(5):754-761.
16 Wu J, Meng Z, Jiang M, et al. Hepatitis B virus suppresses toll-like receptor-mediated innate immune responses in murine parenchymal and nonparenchymal liver cells. Hepatology 2009;49(4):1132-1140.
17 Seglen PO. Preparation of isolated rat liver cells. Methods Cell Biol 1976;13: 29-83.
18 Woo J, Lu L, Rao AS, et al. Isolation, phenotype, and allostimulatory activity of mouse liver dendritic cells. Transplantation 1994;58(4):484-491.
19 Bilsland CA, Diamond MS, Springer TA. The leukocyte integrin p150,95 (CD11c/CD18) as a receptor for iC3b. Activation by a heterologous beta subunit and localization of a ligand recognition site to the I domain.J Immunol 1994;152(9):4582-4589.
20 Lu L, Woo J, Rao AS, et al. Propagation of dendritic cell progenitors from normal mouse liver using granulocyte/macrophage colony-stimulating factor and their maturational development in the presence of type-1 collagen.J Exp Med 1994;179(6):1823-1834.
21 Inaba K, Inaba M, Romani N, et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med 1992;176(6):1693-1702.
22 Son YI, Egawa S, Tatsumi T, et al. A novel bulk-culture method for generating mature dendritic cells from mouse bone marrow cells. J Immunol Methods 2002;262(1-2):145-157.
23 Anders HJ, Zecher D, Pawar RD, et al. Molecular mechanisms of autoimmunity triggered by microbial infection. Arthritis Res Ther 2005;7(5):215-224.
24 Szabo G, Dolganiuc A, Mandrekar P. Pattern recognition receptors:a contemporary view on liver diseases. Hepatology 2006;44(2):287-298.
25 Beutler B. Inferences, questions and possibilities in Toll-like receptor signalling. Nature 2004;430:257-263.
26 Schulz O, Diebold SS, Chen M, et al. Toll-like receptor 3 promotes cross-priming to virus-infected cells. Nature 2005;433:887-892.
27 Matsumoto M, Funami K, Oshiumi H, et al. Toll-like receptor 3:a link between toll-like receptor, interferon and viruses. Microbiol Immunol 2004;48:147-154.
28 Sen GC, Sarkar SN. Transcriptional signaling by double-stranded RNA:role of TLR3. Cytokine Growth Factor Rev 2005; 16:1-14.
29 Sasai M, Shingai M, Funami K, et al. NAK-associated protein 1 participates in both the TLR3 and the cytoplasmic pathways in type I IFN induction.J Immunol 2006;177(12):8676-8683.
1. Medzhitov R, Preston-Hurlburt P,Janeway CA Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature.1997; 388 (6640):394-397
2. Szabo G, Dolganiuc A,Mandrekar P. Pattern recognition receptors:a contemporary view on liver diseases. Hepatology.2006; 44 (2):287-298
3. Anders HJ, Zecher D, Pawar RD, et al. Molecular mechanisms of autoimmunity triggered by microbial infection. Arthritis Res Ther.2005; 7 (5):215-224
4. Isogawa M, Robek MD, Furuichi Y, et al. Toll-like receptor signaling inhibits hepatitis B virus replication in vivo. J Virol.2005; 79 (11):7269-7272
5. Gantner BN, Simmons RM,Canavera SJ Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2. J Exp Med.2003; 197(9):1107-1117
6. Kariko K, Ni H, Capodici J, et al. mRNA is an endogenous ligand for Toll-like receptor 3. J Biol Chem.2004; 279 (13):12542-12550
7. Weber F, Wagner V, Rasmussen SB, et al. Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses.J Virol.2006; 80:5059-5064
8. Wang Z, Xiang L, Shao J, et al. The 31 CCACCA sequence of tRNAAla(UGC) is the motif that is important in inducing Th1-like immune response, and this motif can be recognized by Toll-like receptor 3. Clin Vaccine Immunol.2006; 13 (7):733-739
9. Ito T, Amakawa R,Fukuhara S. Roles of toll-like receptors in natural interferon-producing cells as sensors in immune surveillance. Hum Immunol. 2002; 63 (12):1120-1125
10. Jun Wu, Mengji Lu, Zhongji Meng, et al. Toll-like Receptor-Mediated Control of HBV Replication by Nonparenchymal Liver Cells in Mice. Hepatology.2007; 46:1769-1778
11. Cheng J, Imanishi H, Morisaki H, et al. Recombinant HBsAg inhibits LPS-induced COX-2 expression and IL-18 production by interfering with the NFkappaB pathway in a human monocytic cell line, THP-1. JHepatol.2005; 43 (3):465-471
12. Thompson AJ, Locarnini S, Lau GK, et al. Quantitative HBeAg levels and patterns of TLR2 and TLR4 expression on CD 14+monocytes during potent antiviral therapy for chronic hepatitis B (CHB). Hepatology.2005; 42(Suppl):579A
13. Hui CK, Zhang HY, Lee NP, et al. Upregulation of Toll-like receptor 7 is essential for clearance of hepatitis B surface antigen in chronic hepatitis B virus (HBV) infection. Hepatology.2005; 42 (Suppl):707A-708A
14. Visvanathan K, Skinner NA, Thompson AJ, et al. Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein. Hepatology. 2007;45(1):102-110
15. Chen MQ, Shi GF, Li Q, et al. Study on the expression of toll-like receptor 3 (TLR3) on dendritic cells derived from peripheral blood monocyte of chronic hepatitis B patients. Chinese Journal of Infectious Diseases.2007; 25(12):719-722
2 Liu FM, Zhuang H. Management of hepatitisB in China. Chin Med J 2009; 122: 1-2.
3 Tsai SL, Huang SN. T cell mechanisms in the immunopathogenesis of viral hepatitis B and C. J Gastroenterol Hepatol 1997;12(9-10):S227-235.
4 Hugues S, Boissonnas A, Amigorena S, et al. The dynamics of dendritic cell-T cell interactions in priming and tolerance. Curr Opin Immunol 2006; 18(4):491-495.
5 Ito T, Amakawa R, Fukuhara S. Roles of toll-like receptors in natural interferon-producing cells as sensors in immune surveillance. Hum Immunol 2002;63(12):1120-1125.
6 Beckebaum S, Cicinnati VR, Zhang X, et al. Hepatitis B virus-induced defect of monocyte-derived dendritic cells leads to impaired T helper type 1 response in vitro: mechanisms for viral immune escape. Immunology 2003;109(4):487-495.
7陈明泉,施光峰,卢清等.不同病毒载量的慢性乙型肝炎患者外周血树突状细胞的表型和功能.中华肝脏病杂志2007；15(1)：19-23.
8李谦,陈明泉,李宁等.聚肌胞刺激后慢性乙型肝炎患者树突状细胞内Toll样受体3的表达变化.中华传染病杂志2009；27(12)：733-737.
9张玉杰,施光峰,李谦等.慢性乙型肝炎患者树突状细胞Toll样受体3触发后Ⅰ型干扰素的表达.中华传染病杂志2009；27(6)：343-347.
10安宝燕,谢青,王晖等.干扰素调节因子3在慢性乙型肝炎患者外周血树突状细胞中的表达及意义.世界华人消化杂志2008；16(17)：1873-1879.
11 Seth RB, Sun L, Ea CK, et al. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NFkappaB and IRF 3. Cell 2005;122:669-682.
12 Kawai T, Takahashi K, Sato S, et al. IPS-1, an adaptor triggering RIG-I-and Mda5-mediated type I interferon induction. Nat Immunol 2005;6:981-988.
13 Meylan E, Curran J, Hofmann K, et al. Cardif is an adaptor protein in the RIG-Ⅰ antiviral pathway and is targeted by hepatitis C virus. Nature 2005;437:1167-1172.
14 Hart DN, Fabre JW. Demonstration and characterization of la-positive dendritic cells in the interstitial connective tissues of rat heart and other tissues, but not brain. J Exp Med 1981;154(2):347-361.
15 Prickett TC, McKenzie JL, Hart DN. Characterization of interstitial dendritic cells in human liver. Transplantation 1988;46(5):754-761.
16 Wu J, Meng Z, Jiang M, et al. Hepatitis B virus suppresses toll-like receptor-mediated innate immune responses in murine parenchymal and nonparenchymal liver cells. Hepatology 2009;49(4):1132-1140.
17 Seglen PO. Preparation of isolated rat liver cells. Methods Cell Biol 1976;13: 29-83.
18 Woo J, Lu L, Rao AS, et al. Isolation, phenotype, and allostimulatory activity of mouse liver dendritic cells. Transplantation 1994;58(4):484-491.
19 Bilsland CA, Diamond MS, Springer TA. The leukocyte integrin p150,95 (CD11c/CD18) as a receptor for iC3b. Activation by a heterologous beta subunit and localization of a ligand recognition site to the I domain.J Immunol 1994;152(9):4582-4589.
20 Lu L, Woo J, Rao AS, et al. Propagation of dendritic cell progenitors from normal mouse liver using granulocyte/macrophage colony-stimulating factor and their maturational development in the presence of type-1 collagen.J Exp Med 1994;179(6):1823-1834.
21 Inaba K, Inaba M, Romani N, et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med 1992;176(6):1693-1702.
22 Son YI, Egawa S, Tatsumi T, et al. A novel bulk-culture method for generating mature dendritic cells from mouse bone marrow cells. J Immunol Methods 2002;262(1-2):145-157.
23 Anders HJ, Zecher D, Pawar RD, et al. Molecular mechanisms of autoimmunity triggered by microbial infection. Arthritis Res Ther 2005;7(5):215-224.
24 Szabo G, Dolganiuc A, Mandrekar P. Pattern recognition receptors:a contemporary view on liver diseases. Hepatology 2006;44(2):287-298.
25 Beutler B. Inferences, questions and possibilities in Toll-like receptor signalling. Nature 2004;430:257-263.
26 Schulz O, Diebold SS, Chen M, et al. Toll-like receptor 3 promotes cross-priming to virus-infected cells. Nature 2005;433:887-892.
27 Matsumoto M, Funami K, Oshiumi H, et al. Toll-like receptor 3:a link between toll-like receptor, interferon and viruses. Microbiol Immunol 2004;48:147-154.
28 Sen GC, Sarkar SN. Transcriptional signaling by double-stranded RNA:role of TLR3. Cytokine Growth Factor Rev 2005; 16:1-14.
29 Sasai M, Shingai M, Funami K, et al. NAK-associated protein 1 participates in both the TLR3 and the cytoplasmic pathways in type I IFN induction.J Immunol 2006;177(12):8676-8683.
1. Medzhitov R, Preston-Hurlburt P,Janeway CA Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature.1997; 388 (6640):394-397
2. Szabo G, Dolganiuc A,Mandrekar P. Pattern recognition receptors:a contemporary view on liver diseases. Hepatology.2006; 44 (2):287-298
3. Anders HJ, Zecher D, Pawar RD, et al. Molecular mechanisms of autoimmunity triggered by microbial infection. Arthritis Res Ther.2005; 7 (5):215-224
4. Isogawa M, Robek MD, Furuichi Y, et al. Toll-like receptor signaling inhibits hepatitis B virus replication in vivo. J Virol.2005; 79 (11):7269-7272
5. Gantner BN, Simmons RM,Canavera SJ Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2. J Exp Med.2003; 197(9):1107-1117
6. Kariko K, Ni H, Capodici J, et al. mRNA is an endogenous ligand for Toll-like receptor 3. J Biol Chem.2004; 279 (13):12542-12550
7. Weber F, Wagner V, Rasmussen SB, et al. Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses.J Virol.2006; 80:5059-5064
8. Wang Z, Xiang L, Shao J, et al. The 31 CCACCA sequence of tRNAAla(UGC) is the motif that is important in inducing Th1-like immune response, and this motif can be recognized by Toll-like receptor 3. Clin Vaccine Immunol.2006; 13 (7):733-739
9. Ito T, Amakawa R,Fukuhara S. Roles of toll-like receptors in natural interferon-producing cells as sensors in immune surveillance. Hum Immunol. 2002; 63 (12):1120-1125
10. Jun Wu, Mengji Lu, Zhongji Meng, et al. Toll-like Receptor-Mediated Control of HBV Replication by Nonparenchymal Liver Cells in Mice. Hepatology.2007; 46:1769-1778
11. Cheng J, Imanishi H, Morisaki H, et al. Recombinant HBsAg inhibits LPS-induced COX-2 expression and IL-18 production by interfering with the NFkappaB pathway in a human monocytic cell line, THP-1. JHepatol.2005; 43 (3):465-471
12. Thompson AJ, Locarnini S, Lau GK, et al. Quantitative HBeAg levels and patterns of TLR2 and TLR4 expression on CD 14+monocytes during potent antiviral therapy for chronic hepatitis B (CHB). Hepatology.2005; 42(Suppl):579A
13. Hui CK, Zhang HY, Lee NP, et al. Upregulation of Toll-like receptor 7 is essential for clearance of hepatitis B surface antigen in chronic hepatitis B virus (HBV) infection. Hepatology.2005; 42 (Suppl):707A-708A
14. Visvanathan K, Skinner NA, Thompson AJ, et al. Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein. Hepatology. 2007;45(1):102-110
15. Chen MQ, Shi GF, Li Q, et al. Study on the expression of toll-like receptor 3 (TLR3) on dendritic cells derived from peripheral blood monocyte of chronic hepatitis B patients. Chinese Journal of Infectious Diseases.2007; 25(12):719-722