慢加急性乙型肝炎肝衰竭患者GSTM3基因启动子甲基化状态及氧化损伤分析

发布时间：2018-04-08 09:19

本文选题：慢加急性乙型肝炎肝衰竭　切入点：GSTM3　出处：《山东大学》2013年博士论文

【摘要】：第一部分慢加急性乙型肝炎肝衰竭患者GSTM3基因启动子甲基化状态分析慢加急性肝衰竭(acute-on-chronic liver failure, ACLF)是在慢性肝病基础上出现急性肝功能失代偿的临床表现,主要由感染、酒精、肝毒性药物等因素诱发,临床表现为高胆红素血症、凝血功能障碍,并发腹水、肝性脑病和(或)肝肾综合征以及多脏器功能衰竭等,全身血液动力学改变伴随ACLF的病情进展,其病理生理过程包括全身炎症反应综合征、炎症及氧化应激等,死亡率极高。在我国,引起ACLF的首要病因是乙型肝炎病毒(hepatitis B virus, HBV)感染,由慢性乙型病毒性肝炎所致的ACLF称为慢加急性乙型肝炎肝衰竭(acute-on-chronic hepatitis B liver failure, ACHBLF),是我国最常见的肝衰竭类型,占80%-90%。在临床上ACHBLF缺乏特异、有效的治疗手段,病情凶险,进展迅速,绝大部分患者预后差。ACHBLF的发病机制尚不完全明确。 DNA甲基化是转录水平的DNA修饰方式之一,发生在CpG岛上,它在调节基因表达及维持细胞正常分化中起着重要作用。目前认为DNA甲基化可抑制基因表达。甲基化程度高,基因表达降低。谷胱甘肽-S-转移酶M3(glutathione-S-transferase M3, GSTM3)是谷胱甘肽-S-转移酶(glutathione-S-transferases, GSTs)超家族一种重要的同工酶,具有较强的抗氧化应激功能,是机体内抗氧化防御体系的重要成分之一,具备清除活性氧(或称氧自由基)(reactive oxygen species, ROS)的能力。GSTM3基因启动子发生甲基化时可导致该基因表达下调,造成其抗氧化功能下降,ROS在体内或细胞内蓄积,对肝脏的保护作用降低,可能会加重氧化损伤对肝功能的损害。基因启动子甲基化是目前研究的热点,在恶性疾病的早期诊断、风险评估、早期复发预测、治疗反应监测及预后判断中,异常的DNA甲基化位点已经成为最有前景的分子标志物之一。目前有关GSTs超家族成员GSTM3基因启动子甲基化的研究多侧重于与膀胱癌、肝癌、前列腺癌等肿瘤发生、发展的相关性。我国肝癌患者中约90%有HBV感染背景,但目前尚未见对慢性乙型肝炎(chronic hepatitis B, CHB),特别是重型肝炎患者进行该基因启动子甲基化检测的研究。目的本研究的目的是检测IJACHBLF患者是否存在GSTM3基因启动子区的甲基化异常并通过比较临床预后相关指标,评价其临床意义从而指导临床应用。方法本研究采用病例对照研究设计,病例选自2009年12月至2011年2月在山东大学齐鲁医院住院及门诊就诊患者,其中ACHBLF组30例,CHB组30例；10名健康志愿者作为正常对照组。记录以上研究对象的年龄、性别等指标,收集肝功、肾功、凝血功能、HBV-DNA定量等临床指标并进行比较。用试剂盒提取外周血DNA,并用重亚硫酸盐法对其进行甲基化修饰。应用甲基化特异性聚合酶链反应(methylation-specific polymerase chain reaction, MSP)方法检测三组研究对象GSTM3基因的启动子甲基化状态并进行比较。根据检测结果将ACHBLF组分为甲基化组和非甲基化组,计算各组的终末期肝病模型(model for end-stage liver disease, MELD)积分及死亡率,通过比较进行临床预后评价。所有统计学处理采用统计软件SPSS13.0for Windows分析。组间临床资料比较、MELD积分比较应用独立样本t检验,甲基化率的比较应用卡方检验,死亡率比较应用Fisher's确切概率法。P0.05定为差异有统计学意义。结果 1、ACHBLF组的血清谷丙转氨酶(alanine aminotransferase, ALT)水平(516.110±527.137IU/L)明显高于CHB组(264.633±274.053IU/L,P=0.024),谷草转氨酶(aspartate aminotransferase, AST)水平(396.283±337.062IU／L)明显高于CHB组(212.153±222.816IU／L,P=0.016),总胆红素(total bilirubin, TBIL)水平(383.483±137.034μmol/L)明显高于CHB组(37.400±27.004μmol/L,P0.001),凝血酶原活动度(prothrombin time activity, PTA)水平(31.187±8.013%)明显低于CHB组(90.000±9.766%,P0.001)。 2、30例ACHBLF患者中9例检测至GSTM3基因启动子甲基化,比率为30%,30例CHB患者中2例检测至GSTM3基因启动子甲基化,比率为6.7%,两组比较差异有统计学意义(X2=5.455,P=0.020)。 3、ACHBLF患者甲基化组的血清TBIL水平(527.922±113.564μmol/L)明显高于非甲基化组(321.581±93.048μmol/L,P0.001),PTA水平(24.811±10.697%)明显低于非甲基化组(33.919±4.604%,P=0.035)。 4、ACHBLF患者甲基化组的MELD积分(22.706±2.669)明显高于非甲基化组(18.765±4.808,P=0.029)。ACHBLF患者有8人死亡,死亡率为26.7%,其中5人属于甲基化组,死亡率为55.6%,3人属于非甲基化组,死亡率为14.3%,甲基化组的死亡率明显高于非甲基化组(P=0.032)。结论我们的研究结果首次表明ACHBLF患者存在GSTM3基因启动子甲基化的异常,启动子甲基化可能导致GSTM3基因表达下降,抑制其抗氧化功能,加重氧化损伤程度,在CHB进展为ACHBLF的过程中可能具有重要作用,检测该基因启动子甲基化状态可作为判断ACHBLF患者预后的重要指标。第二部分慢加急性乙型肝炎肝衰竭患者GSTM3基因启动子甲基化状态与氧化应激的相关性分析氧化应激是机体的氧化能力与抗氧化能力失衡,氧化程度超出氧化物质的清除能力,导致活性氧(或称氧自由基)(reactive oxygen species, ROS)在体内或细胞内蓄积而引起的细胞毒性,最终导致组织损伤的过程。氧化应激可直接或间接引起脂质、蛋白质、酶和DNA的氧化或损伤,诱发基因突变、蛋白质变性和脂质过氧化,与肿瘤、心脑血管疾病、糖尿病、各种肝病(包括病毒性肝炎、肝纤维化、肝癌)等多种疾病密切相关。肝脏含有丰富的线粒体,是ROS攻击的主要器官。线粒体呼吸链复合体利用电子传递生产三磷酸腺苷(adenosine triphosphate, ATP),是ROS的主要来源。氧化损伤可以加快肝脏病变的进展。慢加急性肝衰竭(acute-on-chronic liver failure, ACLF)是多种因素参与的复杂过程,炎症和氧化应激在其发生发展中起着重要作用。肝病时患者体内免疫功能紊乱,炎症反应增强,免疫复合物形成,补体活化及胶原纤维形成等均可刺激ROS生成增多,可引起肝细胞过氧化脂质形成。机体内ROS生成与清除处于失平衡状态持续时间越久,组织细胞抗氧化防御保护功能越弱,肝损伤程度越重。肝细胞氧化应激可导致细胞膜脂质过氧化,形成过氧化产物如丙二醛(malondialdehyde, MDA)、酮类、羟氢氧基等。MDA是脂质过氧化的主要代谢产物,是研究脂质过氧化并反映氧化损伤的重要生物标志物,体内MDA水平增高,提示存在氧化损伤；谷胱甘肽-S-转移酶(glutathione-S-transferase, GST)是一种抗氧化酶,它可修复ROS损伤的膜磷脂、抑制微粒体过氧化反应等,通过多种方式起到抗氧化作用。肝细胞受损时GST可以快速释放入血,血清GST活性测定是一项理想的反映肝细胞损害程度的指标,具有良好的特异性和敏感性。二者的水平变化可反映体内自由基产生和清除这一动态平衡过程。谷胱甘肽-S-转移酶M3(glutathione-S-transferase M3, GSTM3)基因表达的改变会影响GST的抗氧化功能。我们第一部分的研究结果首次表明慢加急性乙型肝炎肝衰竭(acute-on-chronic hepatitis B liver failure, ACHBLF)患者存在GSTM3基因启动子甲基化的异常,启动子甲基化可能导致GSTM3基因表达下降,抑制其抗氧化功能,加重氧化损伤程度,在慢性乙型肝炎(chronic hepatitis B, CHB)进展为ACHBLF的过程中可能具有重要作用,检测该基因启动子甲基化可作为判断ACHBLF患者预后的重要指标。目的本研究的目的是通过检测血清MDA和GST表达水平,并与终末期肝病模型(model for end-stage liver disease, MELD)积分进行相关分析,探讨ACHBLF患者GSTM3基因启动子甲基化状态与氧化应激的关系,从而对ACHBLF的病情发展、评估预后及临床治疗提供理论基础。方法应用甲基化特异性聚合酶链反应(methylation-specific polymerase chain reaction, MSP)检测GSTM3基因启动子甲基化状态,具体方法同实验第一部分,应用酶联免疫吸附测定(enzyme-linked immunosorbent assay, ELISA)技术检测CHB患者、ACHBLF患者血清MDA及GST的表达水平,并对ACHBLF患者进行MDA.GST水平与MELD积分的相关性分析。所有统计学处理采用统计软件SPSS13.0for Windows分析。组间MDA及GST水平的比较应用独立样本t检验,应用Spearman's检验进行MDA、GST水平与MELD积分的相关性分析。P0.05定为差异有统计学意义。结果 1、ACHBLF组的血清MDA水平(13.045±5.416pmol/mg)明显高于CHB组(9.387±5.332pmol/mg, P=0.011)。 2、ACHBLF甲基化组的血清MDA水平(16.956±.155pmol/mg)明显高于非甲基化组(11.369±5.553pmol/mg, P0.001)。 3、ACHBLF组的血清GST水平(1318.081±273.887mIU/L)明显高于CHB组(666.832±181.267mIU/L, P0.001)。 4、ACHBLF甲基化组的血清GST水平(1381.678±236.841mIU/L)与非甲基化组相比差异无统计学意义(1262.253±202.782mIU/L, P=0.170)。 5、ACHBLF组的血清MDA水平与MELD积分呈正相关(r=0.588,P=0.001),而血清GST水平与MELD积分无相关性(r=0.115,P=0.546)。结论 ACHBLF患者GSTM3基因启动子甲基化状态与氧化应激存在相关性,该基因启动子甲基化异常参与氧化应激对HBV感染患者的肝功能损害,并且氧化应激损伤程度与患者的病情严重程度成正相关,从而为抗氧化剂治疗ACHBLF提供理论依据。
[Abstract]:Analysis of methylation status of GSTM3 gene promoter in patients with chronic hepatitis B liver failure in the first part

Acute - on - chronic liver failure ( ACLF ) is the main clinical manifestation of acute liver failure on the basis of chronic liver disease , mainly caused by infection , alcohol , hepatotoxic drugs , etc . The clinical manifestations include systemic inflammatory response syndrome , coagulation dysfunction , concurrent ascites , hepatic encephalopathy and / or hepatorenal syndrome as well as multiple organ failure .

DNA methylation is one of the DNA modification methods of transcription level . It plays an important role in regulating gene expression and maintaining normal differentiation of cells .

The methylation of GSTM3 gene promoter is one of the most promising molecular markers in the early diagnosis , risk assessment , early recurrence prediction , therapeutic response monitoring and prognosis of malignant disease .

Purpose

The purpose of this study was to evaluate the presence or absence of methylation abnormalities in the promoter region of the GSTM3 gene in IJACHBLF patients and to evaluate their clinical significance by comparing the clinical outcome - related indicators to guide the clinical application .

method

The case - control study was used in the study , and the cases were selected from December 2009 to February 2011 in the hospital in Qu Hospital of Shandong University and the outpatient visits . Among them , 30 cases were ACHBLF group and 30 patients in the group of the patients in the patients with the patients in the patients with the patients were treated with the treatment group ( 30 cases ) ;
Ten healthy volunteers were used as the normal control group . The age and sex indexes of these subjects were recorded and compared . DNA was extracted from peripheral blood by means of kit . The methylation state of the promoter of GSTM3 gene was detected by methylation - specific polymerase chain reaction ( MSP ) method and compared . The ACHBLF component was used as methylation group and non - methylation group to calculate the integral and mortality rate of the end - stage liver disease ( MELD ) of each group , and the clinical prognosis was evaluated by comparison .

All the statistical treatments were analyzed by SPSS 13.0 for Windows . The comparison of the clinical data between the groups , the comparison of the MELD integral with the independent sample t test , the comparison of methylation rate and the Fisher ' s exact probability method were compared with the Fisher ' s exact probability method ( P0.05 ) .

Results

1 . Serum alanine aminotransferase ( ALT ) level of ACHBLF group ( 516.110 卤 527.137 IU / L ) was significantly higher than that in the control group ( 264.633 卤 274.053 IU / L , P = 0 . 024 ) , and the level of total bilirubin ( TBIL ) ( 383.483 卤 337.062IU / L ) was significantly higher than that in group B ( 37.400 卤 27.004 渭mol / L , P0.001 ) , prothrombin activity ( PTA ) level ( 31.187 卤 8.013 % ) was significantly lower than that in the group B ( 90.000 卤 9.766 % , P0.001 ) .

2 . The methylation of GSTM3 gene was detected in 9 of 30 patients with ACHBLF , the ratio was 30 % , and the methylation of GSTM3 gene promoter was detected in 2 of 30 patients with hepatitis B . The ratio was 6.7 % . There was significant difference between the two groups ( X2 = 5.455 , P = 0.020 ) .

3 . The serum TBIL level of the methylated group of ACHBLF was significantly higher than that in the non - methylated group ( 321.581 卤 93.48 渭mol / L , P0.001 ) , and the PTA level ( 24.811 卤 10.697 % ) was significantly lower than that in the non - methylation group ( 33.919 卤 4.604 % , P = 0.035 ) .

4 . The MELD integral ( 22.706 卤 2.669 ) of the methylated group in ACHBLF was significantly higher than that in the non - methylation group ( 18.765 卤 4.808 , P = 0.029 ) . Eight of ACHBLF patients died and the mortality rate was 26.7 % . Five of them belonged to methylated group , the mortality rate was 55.6 % , the mortality rate was 14.3 % , and the death rate of methylated group was significantly higher than that of non - methylation group ( P = 0.032 ) .

Conclusion

Our results show that the methylation of GSTM3 gene promoter is abnormal in ACHBLF patients . The methylation of promoter may decrease the expression of GSTM3 gene , inhibit its antioxidant function , increase the degree of oxidative damage , and may play an important role in the process of ACHBLF , and the methylation status of the promoter can be used as an important index to judge the prognosis of ACHBLF .

Correlation analysis of methylation status of GSTM3 gene promoter and oxidative stress in patients with chronic hepatitis B hepatic failure in the second part

Oxidative stress is the main organ of ROS attack . Oxidative stress can directly or indirectly cause oxidation or damage of lipid , protein , enzyme and DNA , induce gene mutation , protein denaturation and lipid peroxidation . It is the main organ of ROS attack . Mitochondrial respiratory chain complex uses electron transfer to produce adenosine triphosphate ( ATP ) , which is the main source of ROS . Oxidative stress can accelerate the progression of hepatic lesion .

Chronic liver failure ( ACLF ) plays an important role in the development of acute - on - chronic liver failure ( ACLF ) .

Oxidative stress in liver cells can lead to lipid peroxidation of cell membranes , and form peroxidic products such as malondialdehyde ( MDA ) , ketones , hydroxyhydrophones and the like . MDA is the main metabolite of lipid peroxidation , which is an important biomarker for lipid peroxidation and reflects oxidative damage , and the level of MDA in vivo is increased , suggesting oxidative damage ;
Glutathione - S - transferase ( GST ) is an anti - oxidant enzyme which can repair ROS - damaged membrane phospholipid , inhibit the peroxidation of microsomes and so on . GST can rapidly release blood into blood and serum GST activity is an ideal indicator reflecting the degree of damage of liver cells . The changes of GST activity can reflect the dynamic balance process of free radical generation and elimination in vivo .

The changes of glutathione - S - transferase M3 ( glutathione - S - transferase M3 , GSTM3 ) gene expression can affect the anti - oxidation function of GST . The results of the first part show that the methylation of GSTM3 gene promoter in patients with acute - on - chronic hepatitis B liver failure ( ACHBLF ) is abnormal . The methylation of the promoter may lead to a decrease in the expression of GSTM3 gene , inhibit its anti - oxidation function , increase the degree of oxidative damage , and detect the promoter methylation of the gene as an important index to judge the prognosis of ACHBLF .

Purpose

The aim of this study was to investigate the relationship between the methylation status of GSTM3 gene promoter and oxidative stress in patients with ACHBLF by detecting the level of serum MDA and GST expression , and to explore the relationship between methylation status and oxidative stress in patients with ACHBLF .

method

The methylation state of GSTM3 gene was detected by methylation - specific polymerase chain reaction ( MSP ) , and the methylation status of GSTM3 gene was determined by enzyme - linked immunosorbent assay ( ELISA ) . The level of serum MDA and GST were measured by enzyme - linked immunosorbent assay ( ELISA ) , and the correlation between the level of MDA and GST in patients with ACHBLF was analyzed .

All the statistical treatments were analyzed by SPSS 13.0 for Windows . The comparison of MDA and GST levels between the groups was independent of t - test , and the correlation between MDA and GST levels and the integration of MELD was analyzed by using the spearman ' s test ( P0.05 ) .

Results

1 . The level of serum MDA in ACHBLF group ( 13.45 卤 5.41 pmol / mg ) was significantly higher than that in group B ( 9.387 卤 5.332 pmol / mg , P = 0 . 011 ) .

2 . The serum MDA level ( 16.956 卤 . 155pmol / mg ) in the ACHBLF methylated group was significantly higher than that in the non - methylation group ( 11.369 卤 5.553pmol / mg , P0.001 ) .

3 . The serum GST level of ACHBLF group ( 1318 . 81 卤 273 . 887mIU / L ) was significantly higher than that in group B ( 666.832 卤 181.267mIU / L , P0.001 ) .

4 . The serum GST levels of ACHBLF methylation group ( 131.678 卤 236.841mIU / L ) were not statistically significant ( 1262.253 卤 202.782mIU / L , P = 0.170 ) .

5 . The level of serum MDA in ACHBLF group was positively correlated with the integration of MELD ( r = 0.588 , P = 0.001 ) , and serum GST level was not correlated with MELD integral ( r = 0.115 , P = 0.5546 ) .

Conclusion

The methylation status of the promoter of GSTM3 gene in ACHBLF was correlated with oxidative stress . The methylation of the promoter was involved in oxidative stress on the liver function of patients with HBV infection , and the degree of oxidative stress injury was positively correlated with the severity of the patient ' s disease , thus providing theoretical basis 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】：2013
【分类号】：R575.3;R512.62

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