SOCS1基因与慢加急性乙型肝炎肝衰竭病情评价及激素治疗的研究

发布时间：2018-02-22 21:12

本文关键词： 慢加急性乙型肝炎肝衰竭 SOCS1 甲基化免疫损伤糖皮质激素　出处：《山东大学》2014年博士论文　论文类型：学位论文

【摘要】：中文摘要第一部分SOCSl基因在慢加急性乙型肝炎肝衰竭中的表达及其甲基化状态的检测研究背景慢加急性乙型肝炎肝衰竭(Acute-on-chronic hepatitis B liver failure, ACHBLF)是指由乙型肝炎病毒(Hepatitis B virus, HBV)慢性感染的基础上,出现急性肝功能失代偿,继发严重的肝脏损害的严重症候群。ACHBLF在我国慢性乙型肝炎患者中发病率较高,以凝血功能障碍,黄疸,肝性脑病,腹水为主要表现,最终导致多器官功能障碍综合征,病程凶险死亡率极高。目前,细胞因子在肝衰竭的发病机制中的作用日益受到人们的重视,一方面细胞因子可使淋巴细胞活化,参与免疫反应清除病毒；另一方面细胞因子直接参与和加重肝衰竭的演变过程,引起免疫病理反应导致肝细胞的坏死。细胞因子信号转导抑制因子(Suppressor of cytokine signaling, SOCS)1属于SOCS蛋白家族,是一类由细胞产生并反馈性阻断细胞因子信号转导过程的负性调节因子之一。研究发现SOCS1可通过负反馈环抑制炎症细胞因子信号的传导,在多种免疫反应中发挥重要作用。而HBV可能导致宿主基因甲基化,使SOCS1表达降低,对炎症因子的抑制作用减弱,可能会加重肝脏的炎症损害。研究表明,SOCS1基因的甲基化与慢性病毒性肝炎的炎症、纤维化及抗病毒治疗存在着一定的相关性。但是在慢加急性乙型肝炎肝衰竭中SOCS1基因表达及启动子的甲基化的情况与炎症因子的关系还未有研究。研究目的通过检测ACHBLF患者外周血单个核细胞内SOCS1的mRNA表达情况、启动子区甲基化状态及血浆中相关炎症因子白介素-6(interleukin-6,IL-6)、干扰素-γ(interferon-γ, IFN-γ)和肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)水平。并与慢性乙型病毒性肝炎(CHB)患者及正常人进行比较,分析SOCS1的甲基化与肝脏免疫损伤的关系,并初步探讨其在慢加急性乙型肝炎肝衰竭患者发病机制中的作用及临床相关性。研究方法研究对象为2009年6月到2013年3月问在山东大学齐鲁医院和烟台传染病医院就诊和治疗的60例ACHBLF患者,60例CHB患者,以及30例门诊健康体检者作为对照。ACHBLF患者诊断符合亚太肝脏研究协会年会(APASL)标准。采用实时荧光定量PCR(real-time quantitative PCR, RT-PCR)方法检狈ACHBLF、CHB患者及健康对照外周血单个核细胞中SOCSl的表达情况,并通过甲基化特异性PCR(Methylation-specific PCR, MSP)检测SOCSl启动子区的甲基化状态。应用酶联免疫吸附剂测定(Enzyme linked immunosorbent assay, ELISA)方法检测ACHBLF、CHB患者及健康对照血浆中IL-6,IFN-γ和TNF-α的水平。并将SOCS1表达量IL-6,IFN-γ和TNF-α的水平与病情严重指标总胆红素(Total bilirubin, TBIL)、凝血酶原活动度(Prothrombin activity, PTA)、血清丙氨酸转氨酶(Alanine aminotransferase, ALT)、HBV-DNA及MELD评分进行相关分析。比较不同SOCS1基因启动子去甲基化状态患者临床指标及预后。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。P0.05为差异具有统计学意义。研究结果 1.ACHBLF患者组和CHB患者组血浆IL-6及TNF-α水平明显高于健康对照组(P0.05)；ACHBLF患者组的IL-6及TNF-α水平在三组中最高,明显高于CHB患者组(P0.05)。ACHBLF患者组血浆IFN-γ水平明显高于CHB患者组,但与健康对照组相比无明显统计学差异。 2.ACHBLF及CHB患者的SOCS1mRNA表达量明显高于健康对照组(P0.01)；ACHBLF患者组的SOCS1表达量明显高于CHB患者组(P0.05)。 3.ACHBLF患者细胞因子IL-6/IFN-γ/TNF-α水平与TBIL、ALT及MELD评分呈正相关,与PTA呈负相关,与HBV-DNA载量无明显相关性。 4ACHBLF患者的SOCS1基因mRNA水平与TBIL、ALT成负相关,与PTA成正相关。另夕SOCS1表达量与ACHBLF患者IL-6/IFN-γ/TNF-α水平分别成负相关。与HBV DNA载量及MELD评分无明显相关性。 5ACHBLF患者组的SOCS1基因启动子的甲基化率(35.0%)明显高于CHB患者组(16.7%,χ2=3.52,P=0.003),在30例健康对照中均未发现SOCS1启动子区的甲基化。 6.在ACHBLF及CHB患者患者中,甲基化组的SOCS1基因表达量明显低于非甲基化组。ACHBLF患者SOCS1启动子区甲基化组的IL-6/IFN-γ/TNF-α水平、TBIL和ALT水平明显高于非甲基化组,而PTA水平则明显低于非甲基化组。另外,ACHBLF患者甲基化组的MELD积分及死亡率高于非甲基化组。结论 SOCS1的表达量与患者的病情严重程度成负相关,SOCS1基因启动子区的甲基化可能参与了相关炎症因子对慢加急性乙型肝炎肝衰竭患者肝功能的免疫损害。中文摘要第二部分SOCS1基因甲基化及表达与糖皮质激素治疗慢加急性乙型肝炎肝衰竭的研究研究背景慢加急性肝衰竭的发生不同时相依次经受了免疫损伤、缺血缺氧性损伤和内毒素血症的三重打击,其中炎症因子参与的免疫损伤在肝衰竭发生中起着至关重要的作用。肝病尤其是肝衰竭患者普遍存在着肾上腺功能不全的情况,因此早期使用糖皮质激素是治疗慢加急性肝衰竭的方法之一,近年来随着对糖皮质激素不良反应及并发症防治手段的增强,使用糖皮质激素调节患者机体免疫功能、抑制炎性反应已经成为治疗慢加急性肝衰竭的一种选择。第一部分的研究已经证实了SOCS1基因启动子区的甲基化参与了相关炎症因子对ACHBLF患者的肝功能损害。但ACHBLF患者中糖皮质激素对SOCS1的影响及对患者预后是否有提示作用尚未有研究。既往研究资料证实糖皮质激素在体内、体外环境均能够促进SOCS1基因表达并引起甲基化状态改变。据此我们推测对ACHBLF患者采用糖皮质激素治疗可能影响SOCS1对相关的炎症反应的调控。本研究有助于我们认识糖皮质激素治疗对ACHBLF患者SOCS1的表达及甲基化状态的影响,揭示SOCS1对糖皮质激素治疗ACHBLF的提示作用,为提供临床治疗理论依据和新的途径。研究目的通过研究糖皮质激素治疗对ACHBLF患者SOCS1表达,甲基化状态及相关基因、细胞因子的影响,进一步探讨分析SOCS1表达与糖皮质激素激素治疗预后的相关性。研究方法研究对象为2007年12月到2013年5月间在山东大学齐鲁医院就诊和治疗的ACHBLF患者47例,以及30例门诊健康体检者作为对照。ACHBLF患者诊断符合亚太肝脏研究协会年会(APASL)标准。所有ACHBLF患者确诊后均给予糖皮质激素治疗4周。应用RT-PCR法检钡ACHBLF患者治疗前,治疗第3天及治疗28天后SOCSl及干扰素调节受体(interferon regulatory factors, IRF)-1,趋化因子配体(C-X-C motif ligand, CXCL)9, CXCL10, CXCL11的表达情况,应用ELISA技术检测ACHBLF患者和健康对照血浆中IL-6及TNF-α水平,结合患者临床指标,与MELD评分进行相关分析,用甲基化特异性PCR法(Methylation Specific PCR,MSP)测定SOCSl基因启动子的甲基化状态,分析糖皮质激素对SOCSl基因启动子区甲基化状态的影响,探讨SOCSl表达与糖皮质激素治疗的相关性。统计学分析采用SPSSl3.0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,相关分析采用Pearson线性相关分析。P0.05为差异具有统计学意义。研究结果 1.激素治疗后ACHBLF患者肝性脑病和腹水症状均有明显改善。MELD积分明显下降。 2.激素治疗前,ACHBLF患者SOCS1基因mRNA基因表达水平明显高于健康对照(P0.05)；ACHBLF生存组的SOCS1mRNA表达量明显高于死亡组(P=0.007)。激素治疗第三天,生存组SOCS1表达量上升；治疗第28天,生存组SOCSl基因(?)mRNA表达量明显下降,而死亡组在治疗中及治疗后SOCS1基因mRNA表达量无明显变化。 3.激素治疗前,ACHBLF患者血浆IL-6及TNF-α水平明显高于健康对照(P0.05)；激素治疗后第三天,ACHBLF患者IL-6及TNF-α水平出现明显下降,其中死亡组TNF-α水平明显高于生存组,而两组IL-6水平在治疗第3天无明显差异。激素治疗第28天,生存组IL-6及TNF-α水平均明显低于死亡组(P0.05)。ACHBLF患者中IFN-γ及STATl相关基因表达量与健康对照相比均有不同程度上升,并在激素治疗3天出现明显下降,在第28天下降均超过30%。 4.SOCS1表达水平与MELD积分在激素治疗前、后均存在明显的负相关性。 5.激素治疗前,ACHBLF患者TBIL水平明显高于健康对照(P0.05),PTA水平明显低于健康对照；ACHBLF患者生存组与死亡组TBIL及PTA水平无明显差别(P0.05)。激素治疗第28天,生存组患者TBIL水平明显下降,PTA明显上升；而死亡组无明显改善。在90天随访结束时采用激素治疗的ACHBLF患者死亡率为52.3%。采用小样本生存率估计(Kaplan-Meier法)对最初存在甲基化及无甲基化的患者分别进行生存分析,发现激素治疗能够明显提高SOCS1非甲基化患者的生存率(P0.05)。 6.激素治疗前有34%的ACHBLF患者存在SOCS1基因的甲基化情况,而30例健康对照中均未发现SOCS1启动子区的甲基化。激素治疗第3天MSP检测ACHBLF患者未发现甲基化状态明显改变,激素治疗第28天检测发现,8名最初存在SOCSl基因启动子甲基化的患者未检测至SOCS1的甲基化状态,其中全部6名最初存在甲基化的生存组患者中有5人出现去甲基化,而l0例死亡组患者中仅有3例出现了去甲基化情况。结论慢加急性乙型肝炎肝衰竭患者中,早期应用糖皮质激素能够明显改善患者肝功能,提高SOCS1基因非甲基化者的生存率,而出现SOCS1甲基化的ACHBLF患者对糖皮质激素治疗效果反应较差,死亡率明显高于无甲基化的患者,预后不良。通过检测ACHBLF患者SOCS1基因甲基化状态,或许可以成为使用糖皮质激素治疗ACHBLF筛选条件。
[Abstract]:Expression of SOCSl gene in the first part of Chinese abstract in chronic hepatitis B liver failure and its methylation status
Research background
Chronic liver failure in patients with acute hepatitis B (Acute-on-chronic hepatitis B liver failure, ACHBLF) is defined by the hepatitis B virus (Hepatitis B, virus, HBV) of chronic infection, acute hepatic decompensation, severe.ACHBLF syndrome secondary to severe liver damage in patients with chronic hepatitis B in China in high incidence the blood coagulation dysfunction, jaundice, hepatic encephalopathy, ascites as the main performance, resulting in multiple organ dysfunction syndrome, patients who a high mortality rate. At present, the role of cytokines in the pathogenesis of liver failure has attracted more and more attention on the one hand, cytokines can make lymphocyte activation, the immune response is involved in the clearance of the virus; the evolution of the other on the one hand, cytokines and direct participation of the aggravation of liver failure, caused by the pathological immune response leading to liver cell necrosis.
Suppressor of cytokine signaling (Suppressor of cytokine signaling SOCS, 1) belongs to SOCS protein family, is a kind of feedback produced by cells and blocking negative cytokine signal transduction factor. The study found that SOCS1 can be inhibited by the negative feedback loop of inflammatory cytokine signal transduction, play an important role in variety immune response. HBV may lead to host gene methylation, SOCS1 expression decreased, decreased inhibition of inflammatory factor, inflammation may aggravate the liver damage.
Studies show that inflammation and methylation of SOCS1 gene in chronic viral hepatitis, fibrosis and antiviral therapy, there is a certain correlation. But the relationship in chronic liver failure acute hepatitis B SOCS1 and inflammatory cytokines gene expression and promoter methylation of the not yet studied.
research objective
The expression of ACHBLF was detected by peripheral blood mononuclear cells of patients with SOCS1 in the mRNA, starting state and hypermethylation in plasma inflammatory cytokines interleukin -6 (interleukin-6, IL-6), interferon gamma (interferon- y, IFN- y) and tumor necrosis factor alpha (tumor alpha factor- alpha necrosis, TNF-) level and with chronic hepatitis B (CHB) patients and normal people were compared, analysis of the relationship between SOCS1 methylation and liver immune injury, and investigate the acute on chronic hepatitis B liver failure patients with the pathogenesis and clinical relevance.
research method
The object of the study is from June 2009 to March 2013. In 60 cases of ACHBLF patients in Qilu Hospital of Shandong University and Yantai infectious disease hospital and treatment, 60 cases of CHB patients, and 30 cases of healthy volunteers as control patients with.ACHBLF diagnosis in line with the Asia Pacific Association for the study of the liver (APASL) annual meeting standard. By using real-time quantitative PCR (real-time quantitative PCR, RT-PCR methods both ACHBLF and CHB) patients and healthy controls SOCSl in peripheral blood mononuclear cells of the expression, and by methylation specific PCR (Methylation-specific PCR, MSP SOCSl) to detect the methylation status of the promoter region. The application of enzyme linked immunosorbent assay (Enzyme linked immunosorbent assay, ELISA) method for detection of ACHBLF CHB patients and healthy controls, plasma IL-6, IFN- y and TNF- a level. And the SOCS1 expression of IL-6, IFN- and TNF- level with the severity of gamma alpha severity index, total cholesterol Red pigment (Total bilirubin, TBIL), prothrombin activity (Prothrombin, activity, PTA), serum alanine aminotransferase (Alanine, aminotransferase, ALT), HBV-DNA and MELD score were analyzed. Comparison of SOCS1 gene promoter methylation in patients with clinical parameters and prognosis. The data were analyzed by SPSS13.0 software, the results were standard deviation (x + s) said, the differences between groups using two independent samples t test or chi square test and correlation analysis using Pearson correlation analysis.P0.05 was statistically significant difference.
Research results
1.ACHBLF patients and CHB patients. Plasma IL-6 and TNF- levels were significantly higher than those in healthy control group (P0.05); IL-6 and TNF- levels in patients with ACHBLF group is the highest in the three group, was significantly higher than that in the group of CHB (P0.05).ACHBLF plasma IFN- in patients with gamma levels were significantly higher than that in CHB group, but compared with the healthy control group no statistically significant difference.
The expression of SOCS1mRNA in the patients with 2.ACHBLF and CHB was significantly higher than that in the healthy control group (P0.01), and the SOCS1 expression in the ACHBLF group was significantly higher than that in the CHB group (P0.05).
The level of cytokine IL-6/IFN- gamma /TNF- alpha in patients with 3.ACHBLF was positively correlated with TBIL, ALT and MELD scores, negatively correlated with PTA, and had no significant correlation with HBV-DNA load.
The mRNA level of SOCS1 gene in 4ACHBLF patients was negatively correlated with TBIL and ALT, and positively correlated with PTA. There was a negative correlation between SOCS1 expression level and IL-6/IFN- gamma /TNF- alpha level in ACHBLF patients. There was no significant correlation between IL-6/IFN- level and IL-6/IFN- score.
The methylation rate of SOCS1 gene promoter in 5ACHBLF group (35%) was significantly higher than that in CHB group (16.7%, 2=3.52, P=0.003), and no methylation in SOCS1 promoter region was found in 30 healthy controls.
6. in ACHBLF and CHB patients, the methylation of SOCS1 gene expression group was significantly lower than the non methylation group of.ACHBLF patients with SOCS1 promoter methylation group IL-6/IFN- gamma /TNF- levels, TBIL and ALT were significantly higher than those in non methylation group, while the level of PTA was significantly lower than that of non methylation group. In addition, MELD integral and mortality in patients with ACHBLF methylation group was higher than that in non methylation group.
conclusion
The expression level of SOCS1 is negatively correlated with the severity of the disease. The methylation of SOCS1 promoter region may be involved in the immune damage of inflammatory factors related to liver function in patients with chronic hepatitis B and liver failure.
Study on the methylation and expression of SOCS1 gene in second parts of Chinese abstract and glucocorticoid therapy for chronic hepatitis B liver failure
Research background
Acute on chronic liver failure and the time dependent undergo immune injury, three against ischemia injury and endotoxemia, the immune inflammatory factors involved in the injury plays a crucial role in liver failure. Liver disease especially in patients with liver failure exists in adrenal insufficiency, so early the use of corticosteroids is the treatment of acute on chronic liver failure in recent years, with the increase of glucocorticoid adverse reaction and complication prevention and treatment, the use of corticosteroids and regulating the immune function of patients, inhibiting the inflammatory reaction has become a choice for the treatment of acute on chronic liver failure.
The first part of the study has confirmed that the SOCS1 gene promoter methylation in liver damage related inflammatory factors in the patients with ACHBLF. But the effect of glucocorticoid on patients with ACHBLF and SOCS1 on the prognosis of patients have suggested a role has not been studied. Previous studies have confirmed that glucocorticoids in vivo and in vitro environment can promote the expression of SOCS1 gene and cause the methylation status change. So we presumed that the ACHBLF patients were treated by glucocorticoid therapy may affect SOCS1 on inflammation related regulation. Help our understanding of glucocorticoid expression and methylation status of ACHBLF SOCS1 in this study revealed that the SOCS1 of suggesting the role of sugar corticosteroids in the treatment of ACHBLF, in order to provide theoretical basis for clinical treatment and new ways.
research objective
Objective to investigate the correlation between SOCS1 expression, methylation status and related genes and cytokines in patients with ACHBLF after glucocorticoid treatment, and further explore the correlation between SOCS1 expression and prognosis of glucocorticoid treatment.
research method
The object of the study is from December 2007 to May 2013 in Qilu Hospital of Shandong University and 47 cases of ACHBLF patients, and 30 cases of healthy volunteers as control patients with.ACHBLF diagnosis in line with the Asia Pacific Association for the study of the liver (APASL) annual meeting standard. All ACHBLF patients were treated with glucocorticoid treatment for 4 weeks. The application of RT-PCR method of detecting ACHBLF patients before treatment, after treatment for third days and 28 days after SOCSl and interferon regulatory receptor (interferon regulatory factors, IRF -1), chemokine ligand (C-X-C motif ligand, CXCL 9, CXCL10), the expression of CXCL11, ELISA was used to detect ACHBLF patients and healthy controls IL-6 and serum TNF- level in plasma, combined with clinical indicators patients with MELD score correlation analysis of specific PCR (Methylation Specific PCR, methyl MSP) determination of methylation status of SOCSl gene promoter and analysis of sugar Effect of promoter methylation status of SOCSl gene promoter of the cortical hormone, to investigate the correlation between SOCSl expression and glucocorticoid treatment. The data were analyzed by SPSSl3.0 software, the standard deviation (x + s), t group difference test using two independent sample data, correlation analysis using Pearson.P0.05 linear correlation analysis for the difference was statistically significant.
Research results
After 1. hormone treatment, the symptoms of hepatic encephalopathy and ascites in ACHBLF patients were obviously improved and the.MELD score was obviously decreased.
2. hormone therapy before ACHBLF gene in patients with SOCS1 gene expression level of mRNA was significantly higher than that of healthy controls (P0.05); the expression of ACHBLF in the survival group SOCS1mRNA was significantly higher than that of death group (P=0.007). Hormone therapy for third days, the survival group SOCS1 expression increased; on the twenty-eighth day of treatment, the survival group SOCSl gene (?) the expression of mRNA was significantly decreased however, the death group during treatment and after treatment of SOCS1 gene mRNA expression had no obvious change.
3. hormone therapy before IL-6 and TNF- levels in plasma of patients with ACHBLF was significantly higher than that of healthy controls (P0.05); third days after hormone therapy in patients with ACHBLF, IL-6 and TNF- levels decreased significantly, the death group TNF- levels were significantly higher than the survival group, but the level of IL-6 in the two groups for third days. No significant differences in hormone therapy the twenty-eighth day survival group IL-6 and TNF- alpha levels were significantly lower than the death group (P0.05) and gamma STATl related gene expression of IFN- and healthy controls increased in varying degrees of.ACHBLF patients, and in 3 days of hormone treatment decreased significantly, reduced more than 30%. in the twenty-eighth world
There was a significant negative correlation between the expression level of 4.SOCS1 and the MELD score before the treatment of the hormone.
5. hormones before treatment, the level of TBIL in patients with ACHBLF was significantly higher than that of healthy controls (P0.05), the level of PTA was significantly lower than the healthy control group; no significant difference in survival of patients with ACHBLF and TBIL and PTA level in the death group (P0.05). The twenty-eighth day survival hormone therapy, patients in group TBIL were decreased and PTA increased significantly; and the death group had no obvious improved. At the end of the 90 day follow-up with mortality in patients with hormone therapy for ACHBLF 52.3%. using the small sample survival rate (Kaplan-Meier method) for initial methylation and non methylation were survival analysis, found that hormone therapy can significantly improve the survival rate of SOCS1 methylation in patients (P0.05).
6. hormone therapy before 34% ACHBLF patients have the methylation status of SOCS1 gene, and 30 healthy controls were not found in methylation of SOCS1 promoter region. Hormone therapy third days MSP detection of ACHBLF patients found no methylation changes obviously, hormone therapy twenty-eighth days detected 8 initial SOCSl gene promoter hypermethylation of patients not detected to SOCS1 methylation, in which all 6 initial methylation in patients with survival group 5 showed demethylation, and l0 cases of death in patients with only 3 cases appeared demethylation. Conclusion patients with acute on chronic hepatitis B liver failure in early stage glucocorticoids can significantly improve the liver function of patients, improve the survival rate of SOCS1 gene in non methylation, and SOCS1 methylation of ACHBLF in patients with poor response to glucocorticoid treatment, mortality was significantly higher than those without Patients with methylation have poor prognosis. By detecting the methylation status of the SOCS1 gene in ACHBLF patients, it may be possible to be used as a glucocorticoid for the treatment of ACHBLF.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R512.62;R575.3

【参考文献】