细胞因子对慢性乙肝病情、HBV复制及HBVDNABCP变异的影响研究
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摘要
目的：探讨细胞因子IL-2、IFN-r、IL-4、IL-10对慢性乙肝患者的病情、乙肝病毒(HBV)复制及HBVDNA BCP变异的影响。
方法：采用PCR微板核酸杂交结合ELISA技术检测HBVDNA BCP变异；PCR结合荧光探针体外DNA扩增技术测定HBVDNA含量；用双抗体夹心ELISA法检测IL-2、IFN-r、IL-4和IL-10水平。
结果：(1)根据研究对象病情及预后不同分组进行统计分析发现：慢性重症肝炎组、肝炎后肝硬化组和慢性肝炎组(包括慢肝轻度和中度)病人的IL-2、IFN-r、IL-4和IL-10水平分别为：(单位：pg／ml)87.8±16.9，265.8±228.2，77.7±95.0，109.8±48.4；58.5±20.5，73.6±52.6，66.9±51.8，77.2±33.7和52.4±20.3，32.3±27.5，50.2±35.3，56.9±34.5，其中，慢性重症肝炎组的IL-2、IFN-r和IL-10水平显著高于肝炎后肝硬化和慢性肝炎组病人的水平(P＜0.05)，而IL-4在各组无明显差别(P＞0.05)；死亡病例组和存活病例组的IL-2、IFN-r、IL-4和IL-10的水平分别为：(pg／ml)91.0±19.9，280.7±243.9，86.8±101.6，70.2±49.6和66.0±19.0，93.9±75.9，61.9±50.6，75.1±31.6。其中，IL-2和IFN-r水平死亡病例明显高于存活患者(P＜0.0F)，IL-4和IL-10
水平在两组之间无明显差异O0．0幻；o）朋WM阮P变异组
禾一变异组的 IL－2，IFN－r，IL－4禾 几－10水平分别为：（pg／ml）61．4
士24．7，128．3士107．1，66．3土56．7，76．7士54．5和 67．l土24．3，
33．1士 98．7，59．4上 43．3，72．5 f 37．2，经统计分析各细胞因于在
两组病人之间无明显差别O均川．lX （）患者 HBVDNA水平随看几七
水平的增高而下降，呈明显负相关关系k一0．9722 ； PN．05儿 其余
三种细胞因子与HBVDNA水平未见相关关系。
结论（1）IL十、IFN－r和 IL刁 可能影响慢乙肝病情发展及预后。
Q）机体的免疫压力似乎对HBVDNA BCP变异无明显影响。
＊ 机体的 I卜2水平对 HBV复制具有一定的抑制作用。
Objective: To probe into the influence IL-2, IFN-r, IL-4 and IL-10 on the state of an illness, replication of HBV and occurrence of HBVDNA BCP mutation in patients with chronic hepatitis B.
Methods: Microplate hybridization -ELISA and Fluorescence Quantitative PCR (FQ-PCR) technology were applied to detect HBVDNA BCP mutation and HBVDNA quantity respectively. The level of IL-2, IFN-r, IL-4 and IL-10 in sera was investigated by ELISA method.
Results: (1) The results shown that the level of IL-2, IFN-r, IL-4 and IL-10 in the groups of chronic heavy hepatitis B(CHH), chronic hepatitis B ( including light, mediate degreed ) (CH) and liver cirrhosis (LC) was 87. 8±16. 9, 265.8± 228.2, 77.7±95.0,
109.8±48.4, 58. 5±20. 5, 73.6±52.6, 66.9±51.8, 77. 2±33.7 and 52.4±20.3, 32.3±27.5, 50.2±35.3, 56.9±34.5 respectively; The level of IL-2, IFN-r and IL-10 in CHH group was significant higher than those in CH and LC groups (P<0. 05), but no significant difference was seen in the leverl of IL-4 (P>0. 05). The level of IL-2, IFN-r, IL-4 and IL-10 in the died and survival patients was 91.0 ± 19. 9, 280.7 ± 243.9, 86.8±101.6, 70. 2 ± 49. 6 and 66. 7 ± 19.0, 93.9 ± 75.9, 61.9 ± 50.6, 75. 1 ± 31.6 respectively. The analysis result showed significant difference between the two groups in the level of IL-2 and IFN-r (P<0. 05), but no significant difference was seen in the level of IL-4 and IL-10 (P>0. 05). (2) The level of IL-2, IFN-r, IL-4 and IL-10 in the HBVDNA BCP mutation and no-mutation groups was 61.4 ± 24.7, 128.3 ± 107.1, 66.3 ± 56.7, 76. 7 ± 54. 5 and 67. 1±24. 3, 93.1±98.7, 59.4 ± 43.3, 72. 5 ± 37. 2 respectively, the matched analysis results showed no significant difference between the two groups (P>0. 1). (3) The
quantity of HBVDNA in sera of patients with chronic hepatitis B was lower with the elevation of the IL-2 level, and showed significant relationship (r=-0. 9722, P<0. 05).
Conclusion: (1) The level of IL-2, IFN-r and IL-10 might affect on the development and prognosis of chronic hepatitis B. (2) The immune pressure could be hardly relation to the occurrence of HBVDNA BCP mutation. (3) IL-2 could restrain the replication of hepatitis B virus.
方法：采用PCR微板核酸杂交结合ELISA技术检测HBVDNA BCP变异；PCR结合荧光探针体外DNA扩增技术测定HBVDNA含量；用双抗体夹心ELISA法检测IL-2、IFN-r、IL-4和IL-10水平。
结果：(1)根据研究对象病情及预后不同分组进行统计分析发现：慢性重症肝炎组、肝炎后肝硬化组和慢性肝炎组(包括慢肝轻度和中度)病人的IL-2、IFN-r、IL-4和IL-10水平分别为：(单位：pg／ml)87.8±16.9，265.8±228.2，77.7±95.0，109.8±48.4；58.5±20.5，73.6±52.6，66.9±51.8，77.2±33.7和52.4±20.3，32.3±27.5，50.2±35.3，56.9±34.5，其中，慢性重症肝炎组的IL-2、IFN-r和IL-10水平显著高于肝炎后肝硬化和慢性肝炎组病人的水平(P＜0.05)，而IL-4在各组无明显差别(P＞0.05)；死亡病例组和存活病例组的IL-2、IFN-r、IL-4和IL-10的水平分别为：(pg／ml)91.0±19.9，280.7±243.9，86.8±101.6，70.2±49.6和66.0±19.0，93.9±75.9，61.9±50.6，75.1±31.6。其中，IL-2和IFN-r水平死亡病例明显高于存活患者(P＜0.0F)，IL-4和IL-10
水平在两组之间无明显差异O0．0幻；o）朋WM阮P变异组
禾一变异组的 IL－2，IFN－r，IL－4禾 几－10水平分别为：（pg／ml）61．4
士24．7，128．3士107．1，66．3土56．7，76．7士54．5和 67．l土24．3，
33．1士 98．7，59．4上 43．3，72．5 f 37．2，经统计分析各细胞因于在
两组病人之间无明显差别O均川．lX （）患者 HBVDNA水平随看几七
水平的增高而下降，呈明显负相关关系k一0．9722 ； PN．05儿 其余
三种细胞因子与HBVDNA水平未见相关关系。
结论（1）IL十、IFN－r和 IL刁 可能影响慢乙肝病情发展及预后。
Q）机体的免疫压力似乎对HBVDNA BCP变异无明显影响。
＊ 机体的 I卜2水平对 HBV复制具有一定的抑制作用。
Objective: To probe into the influence IL-2, IFN-r, IL-4 and IL-10 on the state of an illness, replication of HBV and occurrence of HBVDNA BCP mutation in patients with chronic hepatitis B.
Methods: Microplate hybridization -ELISA and Fluorescence Quantitative PCR (FQ-PCR) technology were applied to detect HBVDNA BCP mutation and HBVDNA quantity respectively. The level of IL-2, IFN-r, IL-4 and IL-10 in sera was investigated by ELISA method.
Results: (1) The results shown that the level of IL-2, IFN-r, IL-4 and IL-10 in the groups of chronic heavy hepatitis B(CHH), chronic hepatitis B ( including light, mediate degreed ) (CH) and liver cirrhosis (LC) was 87. 8±16. 9, 265.8± 228.2, 77.7±95.0,
109.8±48.4, 58. 5±20. 5, 73.6±52.6, 66.9±51.8, 77. 2±33.7 and 52.4±20.3, 32.3±27.5, 50.2±35.3, 56.9±34.5 respectively; The level of IL-2, IFN-r and IL-10 in CHH group was significant higher than those in CH and LC groups (P<0. 05), but no significant difference was seen in the leverl of IL-4 (P>0. 05). The level of IL-2, IFN-r, IL-4 and IL-10 in the died and survival patients was 91.0 ± 19. 9, 280.7 ± 243.9, 86.8±101.6, 70. 2 ± 49. 6 and 66. 7 ± 19.0, 93.9 ± 75.9, 61.9 ± 50.6, 75. 1 ± 31.6 respectively. The analysis result showed significant difference between the two groups in the level of IL-2 and IFN-r (P<0. 05), but no significant difference was seen in the level of IL-4 and IL-10 (P>0. 05). (2) The level of IL-2, IFN-r, IL-4 and IL-10 in the HBVDNA BCP mutation and no-mutation groups was 61.4 ± 24.7, 128.3 ± 107.1, 66.3 ± 56.7, 76. 7 ± 54. 5 and 67. 1±24. 3, 93.1±98.7, 59.4 ± 43.3, 72. 5 ± 37. 2 respectively, the matched analysis results showed no significant difference between the two groups (P>0. 1). (3) The
quantity of HBVDNA in sera of patients with chronic hepatitis B was lower with the elevation of the IL-2 level, and showed significant relationship (r=-0. 9722, P<0. 05).
Conclusion: (1) The level of IL-2, IFN-r and IL-10 might affect on the development and prognosis of chronic hepatitis B. (2) The immune pressure could be hardly relation to the occurrence of HBVDNA BCP mutation. (3) IL-2 could restrain the replication of hepatitis B virus.
引文
[1] 侯金林，陈金军，王战会，等。乙型肝炎病毒株X基因／C基因启动子热点变异。中华医学杂志1998；78(2)：107
[2] Buckwold VE, Zhi Chang Xu, Min Chen, et al. Effect of a Naturally Occurring Mutation in the Hepatitis B Virus Basal Core Promoter on Precore Gene Expression and Viral Replication. J Virology 1996; 70(9): 5848
[3] Baumert TF, Rogers SA, Hasegawak, et al. Two Core Promotor Mutations Idemntified in a Hepatitis B Virus Strain Associated With Fulminant Hepatitis Result in Enhanced Viral Replication. J Clin Invest 1996; 98(10):2268
[4] 中华医学会传染病寄生虫病学会修订．病毒性肝炎防治方案(试行)[J]．中华传染病杂志1995，13(4)：241
[5] Abb AK, Murphy KM, Sher A. Funtional driversity of helper T lymphocyte. Nature 1996; 383:787
[6] Bertoletti A, Elios MM, Boni C, et al. Different cytokine profiles, of intraphepatic T cell in chronic hepatitis B and hepatitis C virus
infection. Gastroenterology 1997;112(1):193
[7] FukudaR, Ishimura N, NguygenT X, et al. The expression of IL-2、IL-4 and interon-ganma(IFN-γ) mRNA using liver biopsies at different plase of acute exacerbation of chronic hepatitis B. Clin Exp Immunol 1995;100(3):451
[8] Lee M, Lee SK, Son M, et al. Expression of Th1 and Th2 type cytokines responding to HBsAg and HBxAg in chronic hepatitis B patients. J Korean Med Sci 1999;14(2):175
[9] 姜荣龙，卢桥生，侯金林，等。辅助性T细胞极化群体在慢性乙型肝炎病毒感染中的作用。中华医学杂志2000；80(10)：741
[10] Bochm U, Klamp T, Groot M, et al. Cellular response to interferon-ganma. Annu Rev Immunol 1997;15:749
[11] 甄真，赵彩彦，孙宏勋，等。白细胞介素6在病毒性肝炎免疫发病机理中的意义。中国实验临床免疫学杂志1999；11(6)：27
[12] 刘志红，戴春笋，李恒，等。雷公藤内酯对人近端肾小管上皮细胞单核细胞趋化因子合成的影响。肾脏病与透析，肾移植杂志 2000；9(5)：431
[13] 王岩，马英骥，杨宝山，等。慢性HBV感染者外周血CD4+T细胞内IL-4和IFN-r的表达。解放军医学杂志1999；24(4)：284
[14] 陈金军，侯金林，王战会，等。乙型肝炎病毒C基因启动子及前C变异的动态研究。中华实验和临床病毒学杂志1999；13(1)：57
[15] 侯金林，陈金军，王战会，等。乙型肝炎病毒毒株X基因／C基因启动子热点变异。中华医学杂志1998；78(2)：107
[16] 郭亚兵，戴炜，卢桥生，等。重型乙型肝炎病毒C基因启动子变异。中华传染病杂志 1999；17(1)：108
[17] Erhart A, Reineke U, Blondin D, et al. Mutations of the core promoter and response to interferon treatment in chronic replicative hepatitis B. Hepatoloy 2000;31(3):718
[18] Chan HL, Hussain M, Lok AS, et al. Different hepatitis B virus genotypes are associated with different mutation in the core peomoter and precore regions during hepatitis B e antigen seroconversion. Hepatology 1999;29:976
[19] Melegari M, Scaglioni PP, Wamds JR. Hepatitis B virus mutants associated with 3TC and famciclovir administration are replication defective. Hepatology 1998;27:628
[20] 黄祖瑚，邢益平，刘宁，等。干扰素疗法与HBV前C区点突变关系的初步研究。南京医科大学学报1997；17(4)：309
[21] 骆抗先 乙型肝炎基础和临床。第二版，北京，人民卫生出版社2001 56
[22] Ganem D, Pollack JR, Tavis J. Hepatitis B virus reverse transcriptase and its many roles in hepadnaviral genomic replication. Infect Agents Dis 1994;3(2-3):451
[23] Geneva-popova M, Murdjeve M. Study on proinflammatory cytokines (IL-1 beta, IL-6, TNF-α) and IL-2 in patients with acute hepatitis B .Folia, Med. 1999;4(1):78
[24] Nagarajn K, Naik SK. Chronic hepatitis B virus carrier have low lymphoproliferative responses to HBsAg and reduced interleukin-2synthesis. Indian J Gastroenterol,1998;17(3):83
[25] 黄元成，陈焰，田德英。IFN-α对慢性乙型肝炎患者血清IL-2和SIL-2R水平的影响。临床内科杂志2000；17(6)：362
[26] 赵彩彦，刘金星，汤慧华，等。病毒性肝炎和原发性肝癌患者血清IL-2相关指标的意义。华人消化杂志1998；6(6)：480
[27] 姜荣龙，卢桥生，骆抗先，等。慢性HBV感染者外周血CD4+T细胞内IL-4和IFN-γ表达。解放军医学杂志1999；24(4)：284
[28] 邢同京，章廉，侯金林，等。慢性乙肝患者应答与乙肝病毒感染单个核细胞的关系。第一军医大学学报2000；20(2)：117
[2] Buckwold VE, Zhi Chang Xu, Min Chen, et al. Effect of a Naturally Occurring Mutation in the Hepatitis B Virus Basal Core Promoter on Precore Gene Expression and Viral Replication. J Virology 1996; 70(9): 5848
[3] Baumert TF, Rogers SA, Hasegawak, et al. Two Core Promotor Mutations Idemntified in a Hepatitis B Virus Strain Associated With Fulminant Hepatitis Result in Enhanced Viral Replication. J Clin Invest 1996; 98(10):2268
[4] 中华医学会传染病寄生虫病学会修订．病毒性肝炎防治方案(试行)[J]．中华传染病杂志1995，13(4)：241
[5] Abb AK, Murphy KM, Sher A. Funtional driversity of helper T lymphocyte. Nature 1996; 383:787
[6] Bertoletti A, Elios MM, Boni C, et al. Different cytokine profiles, of intraphepatic T cell in chronic hepatitis B and hepatitis C virus
infection. Gastroenterology 1997;112(1):193
[7] FukudaR, Ishimura N, NguygenT X, et al. The expression of IL-2、IL-4 and interon-ganma(IFN-γ) mRNA using liver biopsies at different plase of acute exacerbation of chronic hepatitis B. Clin Exp Immunol 1995;100(3):451
[8] Lee M, Lee SK, Son M, et al. Expression of Th1 and Th2 type cytokines responding to HBsAg and HBxAg in chronic hepatitis B patients. J Korean Med Sci 1999;14(2):175
[9] 姜荣龙，卢桥生，侯金林，等。辅助性T细胞极化群体在慢性乙型肝炎病毒感染中的作用。中华医学杂志2000；80(10)：741
[10] Bochm U, Klamp T, Groot M, et al. Cellular response to interferon-ganma. Annu Rev Immunol 1997;15:749
[11] 甄真，赵彩彦，孙宏勋，等。白细胞介素6在病毒性肝炎免疫发病机理中的意义。中国实验临床免疫学杂志1999；11(6)：27
[12] 刘志红，戴春笋，李恒，等。雷公藤内酯对人近端肾小管上皮细胞单核细胞趋化因子合成的影响。肾脏病与透析，肾移植杂志 2000；9(5)：431
[13] 王岩，马英骥，杨宝山，等。慢性HBV感染者外周血CD4+T细胞内IL-4和IFN-r的表达。解放军医学杂志1999；24(4)：284
[14] 陈金军，侯金林，王战会，等。乙型肝炎病毒C基因启动子及前C变异的动态研究。中华实验和临床病毒学杂志1999；13(1)：57
[15] 侯金林，陈金军，王战会，等。乙型肝炎病毒毒株X基因／C基因启动子热点变异。中华医学杂志1998；78(2)：107
[16] 郭亚兵，戴炜，卢桥生，等。重型乙型肝炎病毒C基因启动子变异。中华传染病杂志 1999；17(1)：108
[17] Erhart A, Reineke U, Blondin D, et al. Mutations of the core promoter and response to interferon treatment in chronic replicative hepatitis B. Hepatoloy 2000;31(3):718
[18] Chan HL, Hussain M, Lok AS, et al. Different hepatitis B virus genotypes are associated with different mutation in the core peomoter and precore regions during hepatitis B e antigen seroconversion. Hepatology 1999;29:976
[19] Melegari M, Scaglioni PP, Wamds JR. Hepatitis B virus mutants associated with 3TC and famciclovir administration are replication defective. Hepatology 1998;27:628
[20] 黄祖瑚，邢益平，刘宁，等。干扰素疗法与HBV前C区点突变关系的初步研究。南京医科大学学报1997；17(4)：309
[21] 骆抗先 乙型肝炎基础和临床。第二版，北京，人民卫生出版社2001 56
[22] Ganem D, Pollack JR, Tavis J. Hepatitis B virus reverse transcriptase and its many roles in hepadnaviral genomic replication. Infect Agents Dis 1994;3(2-3):451
[23] Geneva-popova M, Murdjeve M. Study on proinflammatory cytokines (IL-1 beta, IL-6, TNF-α) and IL-2 in patients with acute hepatitis B .Folia, Med. 1999;4(1):78
[24] Nagarajn K, Naik SK. Chronic hepatitis B virus carrier have low lymphoproliferative responses to HBsAg and reduced interleukin-2synthesis. Indian J Gastroenterol,1998;17(3):83
[25] 黄元成，陈焰，田德英。IFN-α对慢性乙型肝炎患者血清IL-2和SIL-2R水平的影响。临床内科杂志2000；17(6)：362
[26] 赵彩彦，刘金星，汤慧华，等。病毒性肝炎和原发性肝癌患者血清IL-2相关指标的意义。华人消化杂志1998；6(6)：480
[27] 姜荣龙，卢桥生，骆抗先，等。慢性HBV感染者外周血CD4+T细胞内IL-4和IFN-γ表达。解放军医学杂志1999；24(4)：284
[28] 邢同京，章廉，侯金林，等。慢性乙肝患者应答与乙肝病毒感染单个核细胞的关系。第一军医大学学报2000；20(2)：117