摘要
研究目的
探讨慢加急性重型乙型病毒性肝炎(acute-on-chronic hepatitis B liver failure, ACHBLF)患者血清干扰素γ(IFN-γ)水平及其与病情严重指标的关系,并研究IFN-γ基因启动子甲基化状态对IFN-γ分泌水平的影响,以明确二者在慢加急性重型乙型病毒性肝炎发病机制中的作用。
研究方法
慢加急性重型乙型病毒性肝炎(acute-on-chronic hepatitis B liver failure, ACHBLF)患者20例,慢性乙型肝炎(chronic hepatitis B, CHB)患者15例,为2008年6月至2009年12月在山东大学齐鲁医院肝病科住院和门诊患者。慢加急性重型乙型病毒性肝炎诊断符合2006年《肝衰竭诊疗指南》,慢性乙型病毒性肝炎诊断符合2000年第十次全国病毒性肝炎及肝病学术会议修订的《病毒性肝炎防治方案》中的诊断标准。正常对照10例,来自健康人群志愿者。用酶联免疫吸附法(ELISA)测定研究对象血清中的IFN-γ的水平,并将IFN-γ的水平与病情严重指标TBIL、PTA、ALT、HBV-DNA及MELD评分进行相关分析;用甲基化特异性PCR法(Methylation Specific PCR, MSP)测定IFN-γ基因启动子的甲基化状态,分析甲基化对IFN-γ水平的影响。统计学分析采用SPSS13.0软件包进行,结果用均数±标准差(x±s)表示,组间差异采用两组独立样本资料的t检验或卡方检验,相关分析采用Pearson线性相关分析。P<0.05为差异具有统计学意义。
结果
1. ACHBLF患者组与CHB患者组比较,血清IFN-γ水平明显升高(P<0.05); ACHBLF患者组与健康对照组相比,血清IFN-γ水平明显降低(P<0.05);CHB患者组与健康对照组相比,血清IFN-γ水平明显降低(P<0.05)
2.在ACHBLF患者组中,血清IFN-γ水平与血清TBIL水平呈明显正相关(r=0.804,P<0.01);血清IFN-γ水平与PTA呈明显负相关(r=-0.552,P<0.05);血清IFN-γ水平与ALT水平、HBV-DNA无相关性。
3.在ACHBLF患者组中,血清IFN-γ水平与MELD评分呈明显正相关(r=0.645,P<0.01)
4 ACHBLF患者组与CHB患者组相比,IFN-γ基因启动子甲基化程度明显降低(P<0.05);.ACHBLF患者组与健康对照组相比,IFN-γ基因启动子甲基化程度明显升高(P<0.05)。
5.在ACHBLF患者组及CHB患者组中,甲基化组与非甲基化组相比,血清IFN-γ水平明显降低(P<0.05)。
结论
1.在慢加急性重型乙型病毒性肝炎患者中,血清IFN-γ水平明显高于CHB患者,并且血清IFN-γ水平与血清总胆红素(TBIL)呈明显正相关,与PTA呈明显负相关,与MELD评分呈明显正相关。提示:IFN-γ可能参与慢加急性重型乙型病毒性肝炎的发病,并且可能是慢加急性重型乙型病毒性肝炎患者病情严重程度的指标之
2.慢加急性重型乙型病毒性肝炎患者及慢性乙型肝炎患者的IFN-γ基因启动子的甲基化程度明显高于正常对照者。提示:IFN-γ基因启动子甲基化参与慢性HBV感染的疾病进程。在慢加急性重型乙型病毒性肝炎患者组及慢性乙型病毒性肝炎患者中,甲基化组与非甲基化组相比,血清IFN-γ水平明显降低(P<0.05),而在正常对照组中,甲基化组与非甲基化组相比,血清IFN-γ水平无差别。提示:HBV感染过程中,IFN-γ基因启动子发生甲基化,进而导致IFN-γ分泌减少。
3.ACHBLF患者组与CHB患者组相比,IFN-γ基因启动子甲基化程度明显降低(P<0.05),提示:IFN-γ基因启动子的异常甲基化,可能参与了慢加急性重型乙型病毒性肝炎的发病。
Objective
Interferon-gamma (IFN-γ), one T helper (Th) 1 immune cytokine, is demonstrated to exacerbate liver function in the progression of chronic hepatitis B (CHB). Recently, the mechanism underlining IFN-γmediates acute liver failure on chronic hepatitis b virus infection is still not fully understood. Therefore, this present study was aimed to:(1) determine the serum expression of IFN-γin the patients with acute-on-chronic hepatitis B liver failure (ACHBLF); (2) identify whether the changed IFN-γprofile was associated with its promoter methylation.
Patients and Methods
20 patients with ACHBLF,15 patients with chronic hepatitis B (CHB), and 10 healthy controls were included in our present study. The level of serum IFN-γwas determined by enzyme-linked immunosorbent assay. The methylation of IFN-y promoter of peripheral blood mononuclear cells (PBMCs) was measured by Methylation specific PCR (MSP).
Resutls
The level of IFN-γin patients with ACHBLF (8.47±1.68 pg/ml) was lower than that in the healthy controls (14.65±3.65 pg/ml, p<0.05), but it was higher than the patients with CHB (7.53±2.59 pg/ml, p<0.05). Moreover, the level of IFN-y had positive correlation with TBIL and MELD score, and a negative correlation with PTA in patients with ACHBLF. The percentage of methylation of the IFN-γpromoter gene was higher in the ACHBLF group (60%) as compared to the control group (20%), but it was lower than CHB group(93.3%). The IFN-y level was significantly decreased in methylation group (7.83±1.39 pg/ml) as compared to unmethylaiton group (9.39±1.71 pg/ml) in patients with ACHBLF.
Conclusion
Methylation of IFN-Gamma promoter is associated with decreased level of IFN-Gamma in the patients with acute-on-chronic hepatitis B liver failure
引文
1.中华医学会感染病分会肝衰竭及人工肝学组,中华医学会肝病分会重型肝病与人工肝学组。肝衰竭诊疗指南.中华肝脏病杂志,2006,14(9):643-646。
2. Shiv Kumar Sarin, Ashish Kumar, John A. Almeida, Yogesh Kumar Chawla, Sheung Tat Fan, Hitendra Garg, H. Janaka de Silva, Saeed Sadiq Hamid, Rajiv Jalan, Piyawat Komolmit, George K. Lau, Qing Liu, Kaushal Madan, Rosmawati Mohamed, Qin Ning, Salimur Rahman, Archana Rastogi, Stephen M. Riordan, Puja Sakhuja, Didier Samuel, Samir Shah, Barjesh Chander Sharma, Praveen Sharma, Yasuhiro Takikawa, Babu Ram Thapa, Chun-Tao Wai, Man-Fung Yuen. Acute-on-chronic liver failure:consensus recommendations of the Asian Pacific Association for the study of the liver (APASL). Hepatol Int 2009,3:269-282.
3.医学会肝病学分会中华医学会感染病学分会。慢性乙型肝炎防治指南。中华流行病学杂志,2006,27:79-88。
4. Zou Z, Chen J, Xin S, Xing H, Li B, Li J, et al. Features of onset of chronic severe hepatitis in 520 cases. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 2002,16:322-325.
5. Sen S, Mohensi S, Sjodin L, Cheshire L, Williams R, Bjornsson E, Jalan R:Baseline SOFA score and its lack of early improvement accurately predicts mortality in patients with acute-on-chronic liver failure. Hepatology 2004,40(11):489A.
6. Stauber R, Stadlbauer V, Struber G, Kaufmann P:Evaluation of four prognostic scores in patients with acute-on-chronic liver failure. J Hepatol 2006,44:69-70.
7. Mommeja MH, Mondou E, Blum MR, et al. Serum HBV DNA as a marker of efficafy during therapy for chronic HBV infection:analysis and review of the literature. Hepatology,2003,37(6):1309-1319.
8. Schuurhuis DH, Joan FA, Nagelkerken Bas, et al. Antigen-antibody immune compelexes empower dendritic cell to efficiently prime specific CD8+ CTL responses in vivo.J Immunol,2002,168(8):2240-2246.
9. Nagaki M, TanaKa M, Sugiyama A,et al.Interlerkin-10 inhibits hepatic injury and tumor necrosis factor-α and interferon-γ expression induced by staphylococcal enterotoxin B or liopolysaccharite in galactosamine-senstitized mice. J Hepatol,1999,31:815-824
10.Minoru Ayada, Tetsuya Ishikawa, Akihiko Okumura et al. Alteration of serum cytokine balances among different phases of chronic hepatitis B virus infection. Hepatology Research.2006,34:214-221.
11.Cheng JY, Cho SW, et al. A ssociation between chronic hepatitis B virus infection and interleukin-10, tumor necrosis factor algha gene Promoter Polymorphisms. Journal of Gastroenterology and Hepatology,2006,21:1163-1169.
12.Ando K, Moriyama T, Guidottl LG, et. Mechanisms of class I restricted immunopathology. A transgenic mouse model of fulminant hepatitis. J Exp Med,1973,178(8); 1541-1554.
13.Leifeld L, Cheng S, Ramakers J, et al.Imbalanced intrahepatic expression of interlerkin 12, interferon gamma, and interleukin 10 in fulminant hepatitis B. Hepatology,2002,36(4):1001-1008.
14.Chen A, Wang L, Zhang JB, et al.H-2 Kd-restricted hepatitis B virus-derived epitope whose specific CD8+ T lymophocytes can produce gamma interferon without cytotoxicity. j. Virol, 2005,79:5568-5576.
15.Wasmuth HE,Kunz D,Yagmur E,et al.Patients with acute on chronic liver failure display'sepsis-like'immune paralysis. J of Hepatol,2005,42;195-201.
16. Wilson CB, Makar KW, Shnyreva M, Fitzpatrick DR. DNA methylation and the expanding epigenetics of T cell lineage commitment. Semin Immunol.2005,17:105-119.
17.Fitzpatrick DR, Wilson CB. Methylation and demethylation in the regulation of genes, cells, and responses in the immune system. Clin Immunol,2003,109:37-45.
18.Young HA, Ghosh P, Ye J, Lederer J, Lichtman A, Gerard JR, et al. Differentiation of the T helper phenotypes by analysis of the methylation state of the IFN-gamma gene. J Immunol, 1994,153:3603-3610.
19.Nam-Hee Kwon, Jin-Seuk Kim, Jin-Young Lee, Mi-Jung Oh, Dong-Chull Choi. DNA methylation and the expression of IL-4 and IFN-y promoter genes in patients with bronchial asthma. J Clin Immunol,2008,28:139-146.
20.中华医学会传染病与寄生虫病学分会、肝病学分会,病毒性肝炎防治方案。中华传染病杂志,2001,19:56-62.
21.Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis. Annu Rev Immunol,1995,13:29-60.
22.Thimme R, Wieland S, Steiger C, et al.CD8+ T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection. J Virol,2003;77(1):68-76.
23.Mosmann TR, Cerwinski H, Bond MW, et al. Two types of murine helper clone definition according to profiles of lymghokine activities and secreted proteins. J Immunol,1986; 136(7):2348-2357.
24.Agrawal S, Agrawal A, Doughty B, et al. Cutting Edge:different toil-like receptor agonists instruct dendritic cells to induce distinct Th responses via differential modulation of extracellular signal-regulated kinase mitogen-acticated protein kinase and c Fos, J Immunol, 2003,171:4984-4989.
25.Isaacs A, Lindenmann J. Virus interference the interferon. Proc R Soc Lond B Biol Sci,1957,147:258-267.
26.Narayan R,Buronfosse T, Schultz U, et al. Rise in gamma interferon expression during resolution of duck hepatits B virus infection. J Gen Virol,2006,87(6):3225-3232.
27.Bertoletti A, Gehring AJ. The immune pesponse during hepatitis B virus infection. J Gen Virol,2006,87(6):1439-1449.
28.Ohta A, Sekimoto M, Sato M, et al. Indispensable role of TNF-alpha and IFN-gamma at the effector hpase of liver injury mediated by Thl cells specific to hepatitis B virus surface antigen. J Immunol,2000; 165(2):956-961.
29.Nishimura T, Ohta A. A critical role for antigen-specific Thl cells in acute liver injury in mice. J Immunol,1999,162(11):6503-6509.
30.Schumann J, Wolf D, Pathl A, et al. Importance of Kupffer cells for T-cell-dependent liver injury in mice. Am J Pathol,2000,157(5): 1671-1683.
31.Ohta S, Nakamuta M, Fukushima M, et al. Beraprost sodium, a prostacyclin (PGI) analogue, ameliorates concanavalin A-induced liver injury in mice. Liver Int,2005,25(5):1061-1068.
32.1shida Y, Konda T, Tsuneyama K, et al. The pathogenic roles of tumor necrosis factor receptor p55 in acetaminophen-induced liver injury in mice. J Leukoc Biol,2004,75(1):59-67.
33.Kato M, Ikeda N, Mdtsushita E, et al.Involvement of IL-10,an anti-inflammatory cytokine in murine liver injury induced by concanavalin A. Hepatol Res,2001,20(2):232-243.
34.姜荣龙,卢桥生,骆抗先,等。慢性乙型病毒性感染者外周血T辅助细胞1、2型因子的表达。中华传染病杂志,2000,18:26-28。
35.Milich DR, Chodel FS, Hughes JL, et al. The hepatitis B virus core and e antigens elicit differert Th cell subsets:antigen structure affect Th cell phenotype. J Virol,1997,71:2192-2201.
36.孙颖,李保森,徐东平,等。肝内IFN-γ及其分泌细胞在乙型慢加急性肝衰竭发病机制中的作用。传染病信息,2008,21(4):227-230。
37.Jian Ge, Kai Wang, Qing-Hua Meng, Zhao-Xia Qi, Fan-Li Meng, Yu-Chen Fan. Implication of Th17 and Thl cells in patients with chronic active hepatitis B. J Clin Immunol.2010,30(1):60-67.
38.Yuki Yasumi, Yasuhiro Takikawa, Ryujin Endo, and Kazuyuki Suzuki. Interleukin-17 as a new marker of severtity of acute hepatic injury. Hepatology Research.2007,37:248-254.
39.Kimura K, Ando K,Tomita E, et al. Elevated intracellular IFN-gamma levels in circulating CD8+ lymphocytes in patients with fulminant hepatitis. Hepatol,1999,31(4):579-583.
40.Hansen TW. Mechanisms of bilirubin toxicity:clinical implications. Clin Perinatol,2002,29:767-778.
41.Hansen TW. Bilirubin brain toxicity. J Perinatol.2001,21(1):48-51.
42.徐道振主编,病毒性肝炎临床实践[M]。北京:人民卫生出版社, 2006,239-314。
43.黄力毅,张丽生,玉艳红,吴继周,黄璐。乙型肝炎病毒核心基因启动子变异与患者血清病毒载量和标志物及肝功能的关系。中华医院感染学杂志,2007,17(12):1481-1484。
44.任金才,李丽君,吴月平。47例慢性乙型肝炎HBV-BCP变异及肝功能、PTA、血清ChE检测临床意义。交通医学,2002,16(4):329-330。
45.Limdi JK, Hyde GM. Evaluation of abnormal liver function testsl. Postgrad Med J,2003,79(932):307-312.
46.杨绍基主编,传染病学[M]。北京:人民卫生出版社,2002,22-41。
47.Crispe IN, Dao T, Klugewitz K, et al. The liver as a site of T cell apoptosis:graveyard, of killing fiele. Immunlo Rev,2000,47-62.
48.Malinchoc M, Kamath PS, Gordon FD, Peine CJ, et al. Amodel to predict poor survival in patients undergoing transjugular intrahepatic portosystemic shunts. Hepatology,2000,31:864-871.
49.Kamath PS, Wiesner RH, Malinhoc M, et al. A model predicts survival in patients with end-stage liver disease. Hepatology,2001, 33:464-470.
50.Edwards EB, Haper AM. Application of a continuous disease severity score to the OPTN liver waiting list. Clin Transpl,2001,9-24.
51.2004 AASLD/ILTS transplant course. Indications for liver transplantation in the MELD era:Evidence-based ptiern seletion. Liver Transpl,2004,10(2):81-92.
52.Wiesner RH. Evidence-base devolution of the MELD/PELD liver allocation policy. Liver Transpl,2005,11 (3):261-263.
53.Said A, Williams J, Holden J, et al. Model for end stage liver diseases core predicts mortality across a broad spectrum of liver disease. J Hepatol,2004,40(6):897-903.
54.马慧,王豪,孙焱,等,失代偿期肝硬化患者的终末期肝病模型预后分析。中华肝脏病杂志,2005,13(6):407-409。
55.Weng ZH, Cai SQ. Analysis of prognosis on patients with severe viral hepatitis using the model for end-stage liver disease. World J Gastroenterol,2005,11(6):899-902.
56.Wolffe AP. Epigenetics:regulation through repression. Science,1999, 286:481-486.
57.Antequera F, Bird A. Number of CpG islands and genes in human and mouse. Proc Natl Acad Set USA,1993,90:11995-11999.
58.Jones PA, Takai D. The role of DNA methylation in mammalian epigenetics. Science,2001,293(5532):1068-1070.
59.Bird AP. CpG-rich-island, and the fuction of DNA methylation. Nature, 1986,321(15):209-213.
60.Robertson KD.DNA methylation, methyltransferases, and cancer. Oncogene,2001,20:3139-3155.
61.Robertson KD, Jones PA. DNA methylaiton:past, present and future directions. Carcinogenesis,2000,21 (3):461-467.
62.Plass C. Cancer epigenomics. Hum Mol Genet,2002,11:2479-2488.
63. Vertino PM, Yen RW, Gao J, et al. De novo methylation of CpG island sequences in human fibroblasts overexpressing DNA(cytosine-5) methyltransferasel. Mol Cell Biol.1996,16(8):4555-4560.
64.Narimatsu T, TamoriA, Koh N, et al. p16 promoter hypermethylation in human hepatocellular carcinoma with or without hepatitis virus infection. Intervirology,2004,47:26-31.
65.Arai M, Yokosuka O, Hirasawa Y, et al. Sequential gene expression changes in cancer cell lines after treatment with the demethylation agent 5-Aza-2'-deoxycytidine. Cancer,2006,106:2514-2525.
66.Jin B, Park DW, Nam KW, et al. CpG methylation of the mouse CYP1A2 promoter. Toxicol Lett,2004,152:11-18.
67.Schmelz K, Wagner M, Dorken B, et al.5-Aza-2'-deoxycytidine induces p21WAF expression by demethylation of p73 leading to p53-independent apoptosis in myeloid leukemia. Int J Cancer,2005, 114:683-695.
68.Jones PA. DNA methylation erros and Cancer. Cancer Res,1996,56: 2463-2467.
69.Lee GR,Kim ST,Spiliangakis CG,et al. T helper cell differentiation: regulation by cis elements and epigenetics. Immunity,2006, 24(4):369-379.
70.Yano S, Ghosh P, Kusaba H, et al. Effect of promoter methylation on the regulation of IFN-gamma gene during in vitro differentiation of human peripheral blood T cells into a Th2 population. J Immunol, 2003,171:2510-2516.
71.Morinobu A, Kanno Y, O'Shea JJ. Discrete roles for histone acetylation in human T helper 1 cell-specific gene expression. J Biol Chem,2004,279:40640-40646.
72.Ta.kaaki Kaneko, Hiroyuki Hosokawa, Masakatsu Yamashita, et al. Chromatin remodeling at the Th2 cytokine gene loci in human type 2 helper T cells. Molecular Immunology,2007,44:2249-2256.
73.Perumal Vivekanandan, Hubert Darius-J Daniel, Rajesh Kannangai, Francisco Martinez-Murillo, and Michael Torbenson. Hepatitis B Viral Replication Induces Methylation of Both Host and Viral DNA. J. Virol. doi:10.1128/JVI.02280-09.
74.Kaneto H, Sasaki S, Yamamoto H, et al. Detection of hypermethylation of the p16 (NK4A) gene promoter in chronic hepatitis and cirrhosis associated with hepatitis B or C virus. Gut, 2001,48:372-377.
75.Tannapfel A. Methylation and other new concepts for the origin of hepatocellular carcinoma. Pathologe,2006,27:284-288.
76.Melvin AJ, McGurn ME, Bort SJ, et al. Hypomethylation of the interferon-gamma gene correlates with its expression by primary T-lineage cells. Eur J I mmunol,1995,25:426-430.
77.Tate PH, Bird AP. Effects of DNA methylation on DNA-bingding proteins and gene expression. Curr Opin Genet Dev.1993, 3(2):226-231.
78.Rountree MR, Selker EU. DNA methylation inhibits elongation but not initiation of transcription in Nerospora crassa.Genes Dev.1997, 11(80):2383-2395.