慢加急性重型乙型病毒性肝炎患者血清IFN-γ水平及其启动子甲基化状态研究
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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
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
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