摘要
慢加急性肝衰竭(Acute on chronic liver failure, ACLF)是指在慢性肝病基础上,短期内发生急性肝功能失代偿的主要临床表现。常见的病因包括(1)感染因素：嗜肝或非嗜肝病毒、乙型肝炎(显性或隐性)或丙型肝炎再活动；(2)非感染因素：有饮酒史、肝毒性药物(中草药)、手术以及自身免疫性肝病等。在我国,慢加急性肝衰竭主要发生于慢性乙型病毒性肝炎(Chronic hepatitis B, CHB)基础上。同时,乙肝病毒(Hepatitis B virus, HBV)感染又是原发性肝癌(hepatocellular carcinoma, HCC)最主要的致病因素,HCC的发生发展的分子机制涉及癌基因、抑癌基因、生长因子、转移相关基因及其受体等多个环节,抑癌基因的表达降低参与了HCC的发生。
谷胱甘肽巯基转移酶PI (Glutathione S-transferase PI, GSTP1)是人体内生物转化最重要的Ⅱ相代谢酶之一,是细胞抗氧化损伤、抗癌变的主要解毒酶。GSTP1属于谷胱甘肽巯基转移酶(Glutathione Stransferase, GST)家族,该家族是还原型谷胱甘肽(reduced glutathione,GSH)发挥对抗脂质过氧化、保护肝细胞膜、清除氧自由基和促进肝脏合成功能等作用时必需的一组催化酶。GSTP1基因启动子区发生甲基化时,会使GSTP1的表达降低,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。
本论文通过检测ACLF患者外周血中的GSTP1启动子区甲基化情况和氧化损伤状态以及肝癌患者的GSTP1蛋白和mRNA表达情况.来研究GSTP1在ACLF和HCC发生中所发挥的作用。
中文摘要第一部分
谷胱甘肽巯基转移酶P1启动子区甲基化与慢加急性肝衰竭患者氧化损伤的相关性研究
肝衰竭是多种因素引起的严重肝脏损害,导致其合成、解毒、排泄和生物转化等功能发生严重障碍或失代偿,出现以凝血机制障碍和黄疸、肝性脑病、腹水等为主要表现的一组临床症候群。根据病理组织学特征和病情发展速度,肝衰竭可被分为四类：急性肝衰竭(acute liver failure, ALF)、亚急性肝衰竭(subacute liver failure, SA)、慢加急性肝衰竭Acute on chronic liver failure, ACLF)和慢性肝衰竭(chronic liver failure, CLF)。慢加急性肝衰竭是指在慢性肝病基础上,短期内发生急性肝功能失代偿的主要临床表现。常见的病因包括感染因素：嗜肝或非嗜肝病毒、乙型肝炎(显性或隐性)或丙型肝炎再活动；非感染因素：有饮酒史、肝毒性药物(中草药)、手术以及自身免疫性肝病等。在我国,慢加急性肝衰竭主要发生于慢性乙型病毒性肝炎(Chronic hepatitis B, CHB)患者。除病毒性肝炎外,常见的肝衰竭病因还有妊娠急性脂肪肝、药物性肝病和酒精性肝病。
谷胱甘肽毓基转移酶P1(Glutathione S-transferase P1，GSTP1)是人体内生物转化最重要的Ⅱ相代谢酶之一,是细胞抗氧化损伤、抗癌变的主要解毒酶。GSTP1属于谷胱甘肽巯基转移酶(GST)家族,该家族是还原型谷胱甘肽(GSH)发挥对抗脂质过氧化、保护肝细胞膜、清除氧自由基和促进肝脏合成功能等作用时必需的一组催化酶。GSTP1基因启动子区发生甲基化时,会使GSTP1的表达降低,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。GSTP1基因启动子甲基化在各种肿瘤中研究较多,如肝癌,前列腺癌等。但慢加急性肝衰竭患者中是否存在GSTP1启动子区甲基化的情况及其与氧化损伤的相关性尚未有研究。
目的
在本研究中,我们致力于分析乙肝病毒感染相关的ACLF患者的GSTP1基因启动子区甲基化状态和氧化损伤水平,并与慢性乙型病毒性肝炎患者进行比较,观察GSTP1甲基化是否ACLF的发生及氧化损伤水平具有相关性以及评价它们的相关性。
方法
1.收集10例正常人、48例ACLF患者和48例CHB患者外周血5ml,密度梯度离心法分离外周血单个核细胞和血清,用试剂盒提取单个核细胞中的基因组DNA。
2.DNA甲基化修饰试剂盒处理DNA将DNA分子中的未甲基化的胞嘧啶(C)转变为尿嘧啶(U)。甲基化特异性PCR(MSP-PCR)法检测外周血中GSTP1基因启动子区的甲基化情况。采用文献的引物并采用其PCR条件,凝胶电泳检测所得产物。
3.采用逆转录实时荧光定量PCR(Real-time fluorescence quantitative PCR, QRT-PCR)的方法来检测ACLF患者和CHB患者外周血中的GSTP1的mRNA表达水平。采用酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA)来测定HCC患者和CHB患者血清中的丙二醛(MDA)和还原型谷胱甘肽(GSH)含量,其中,MDA的血清水平含量代表氧化损伤的水平,GSH的水平代表患者血清的抗氧化损伤水平。
结果
48例ACLF患者中有13例,48例CHB患者中有4例(8.57%),检测至GSTP1甲基化,差别有统计学意义(χ2=5.79,P=0.03),提示GSTP1甲基化可能参与CHB患者进展为ACLF。 ACLF患者外周血单个核细胞中的GSTP1mRNA表达水平较CHB患者的表达水平明显降低(P<0.01)。ACLF患者中GSTP1甲基化组的GSTP1mRNA表达水平较非甲基化组明显降低(P=0.01)。ACLF患者中GSTP1甲基化组和非甲基化组之间的临床指标如ALT、AST、PTA之间没有统计学差异(P>0.05),但甲基化组的TBIL水平明显高于非甲基化组(P=0.04)。ACLF患者血清中的MDA水平较CHB患者的水平明显升高(0.68±0.3ng/ml vs0.42±0.18ng/ml, P=0.02), ACLF患者血清中的GSH的水平较CHB患者的水平明显降低(0.68±0.3ng/ml vs0.42±0.18ng/ml, P=0.02)。 ACLF患者中GSTP1甲基化组的MDA水平也明显高于非甲基化组(14.45±6.23pmol/mg vs11.76±3.94pmol/mg, P=0.04),两组患者的GSH水平没有统计学上的差异。ACLF患者中GSTP1甲基化组和非甲基化组之间的病情程度(MELD积分)没有显著差别(20.32±4.72vs18.57±5.19, P=0.35)。 MDA水平与MELD积分之间存在正相关(r=0.579,P<0.01),即氧化损伤程度与ACLF患者的病情严重程度成正相关。
结论
GSTP1基因启动子区的异常甲基化参与了氧化损伤对ACLF患者的肝功能损害,并且氧化损伤程度与患者的病情严重程度成正相关。
中文摘要第二部分
肝癌患者与慢性乙型病毒性肝炎患者外周血中的GSTP1表达水平和氧化损伤状态的比较
乙型肝炎病毒(Hepatitis B virus, HBV)感染与急慢性肝炎、肝硬化和原发性肝细胞癌的发生和发展密切相关。我国的原发性肝癌(hepatocellular carcinoma, HCC)主要发生于慢性乙型病毒性肝炎(chronic hepatitis B, CHB)的基础上。肝癌的发生发展的分子机制涉及癌基因、抑癌基因、生长因子、转移相关基因及其受体等多个环节。既往多项研究显示肝癌的发生与某些抑癌基因的启动子区甲基化相关,如凋亡相关基因、p16基因、肝细胞生长因子激活剂抑制因子等,这些基因启动子区的甲基化会导致抑癌基因的表达减少,从而促进肝癌的发生和进展。
谷胱甘肽筑基转移酶P1(GSTP1)是人体内生物转化最重要的Ⅱ相代谢酶之一,是细胞抗损伤、抗癌变的主要解毒系统。以往的研究表明多种肿瘤患者体内GSTP1的表达水平存在明显异常。例如,在前列腺癌、胃癌以及肝细胞肝癌中研究者发现存在GSTP1的启动子区甲基化,由基因启动子区甲基化导致的表达量减少可能参与了上述肿瘤的发生,但GSTP1蛋白表达水平的改变以及肝癌患者的氧化损伤状态研究较少。
目的
在本研究中,我们致力于分析乙肝病毒相关的肝癌患者的GSTP1蛋白表达水平、mRNA表达水平以及氧化损伤状态,并与慢性乙型病毒性肝炎患者进行比较,观察GSTP1的表达水平以及氧化损伤状态是否与肝癌的发生具有相关性。
方法
我们首次采用流式细胞仪检测的方法来测定38例乙肝病毒相关肝癌(HCC)患者和38例CHB患者以及10名健康对照者外周血中的GSTP1的表达水平。采用逆转录实时荧光定量PCR(Real-time fluorescence quantitative PCR, QRT-PCR)的方法来检测HCC患者和CHB患者外周血中的GSTP1的nRNA表达水平。采用酶联免疫吸附法(Enzyme linked immunosorbent assay, ELISA)来测定HCC患者和CHB患者血清中的丙二醛(MDA)和黄嘌呤氧化酶(XOD),还原型谷胱甘肽(GSH)以及谷胱甘肽巯基转移酶(GST)含量,其中,MDA和XOD的血清水平含量代表氧化损伤的水平,GSH和GST的水平代表患者血清的抗氧化损伤水平。
结果
乙肝病毒相关的HCC患者外周血中的GSTP1蛋白表达水平较CHB患者水平明显降低(阳性百分率1.13士0.55%vs2.72±1.06%,P=0.005；平均荧光强度2.63±1.02vs5.63±2.33, P=0.002)。与GSTP1蛋白表达水平的变化相似,HCC患者外周血单个核细胞中的GSTP1的mRNA表达水平较CHB患者的表达水平明显降低(中位数：0.04；范围：0.005-0.33vs.中位数：0.06；范围：0.02-1.05；P=0.01)。HCC患者血清中的MDA和XOD水平较CHB患者的水平明显升高(19.13±2.01pmol/mg vs12.99±3.55pmol/mg, P=0.006;183.23±78.09mIU/ml vs53.96±29.28mIU/ml, P<0.01), HCC患者血清中的GSH和GST的水平较CHB患者的水平明显降低(2.81±1.40ng/ml vs5.79±3.03ng/ml, P<0.01;0.26±0.12μg/ml vs1.10±0.56μg/ml, P<0.01)。
结论
HCC患者的GSTP1的蛋白和mRNA表达水平较CHB患者明显降低,GSTP1表达水平的变化可能参与了患者从CHB到HCC的进展,HCC患者的氧化损伤水平较CHB明显升高,GSTP1的表达水平降低可能与肝癌的氧化损伤水平较高相关。
Acute-on-chronic liver failure is a serious syndrome characterized by hepatic decompensation with a short onset of jaundice, very high alanine aminotransferase (ALT) level and low prothrombin time activity (PTa) level, which is an acute deterioration of unknown or known chronic liver disease. In China, it often occurs in chronic hepatitis B patients and diagnosed as acute-on-chronic hepatitis B liver failure (ACLF) according to a recent Asia-Pacific consensus recommendation. As a major cause of cancer-related death worldwide, HCC is one of the most frequent human cancers and demonstrates geogrphic variation, particularly in China.
GST enzymes play a critical role in the protection against oxidative stress, due to that they can utilize various products of oxidative stress as substrates in liver. The enzyme family is composed of alpha, kappa, mu, omega, pi, sigma, theta, and zeta isoforms in humans,. As a member of the enzyme family, GSTP1is expressed in various normal tissues at different levels and its aberrant activity and expression due to the promoter methylation has been demonstrated in many tumors and other diseases.
The present study was designed to evaluate the status of GSTP1promoter methylation, the possible role of GSTP1expression change and redox state in ACLF patients, as well as the potential associations between GSTP1and oxidative stress in HCC patients.
英文摘要第一部分
PROMOTER METHYLATION OF OXIDATIVE-STRESS RELATED ENZYME GSTP1AND OXIDATIVE STRESS ANALYSIS IN ACUTE-ON-CHRONIC HEPATITIS B LIVER FAILURE
Background As an antioxidant enzyme, aberrant promoter methylation of Glutathione-S-transferases PI (GSTP1) may be involved in liver damage caused by oxidative stress in acute-on-chronic hepatitis B liver failure(ACLF).
Objective To explore GSTP1promoter methylation status and oxidative stress of ACLF patients.
Methods DNA was extracted from peripheral blood mononuclear cells (PBMCs) of, chronic hepatitis B (CHB) patients, and normal controls, followed by sodium-bisulfite treatment and methylation-specific PCR (MSP) analysis. Plasma Malondialdehyde (MDA) adduct and was detected by ELISA as oxidative stress marker. Model for end-stage liver disease (MELD) was employed to estimate ACLF severity. GSTP1promoter methylation status of ACLF, chronic hepatitis B (CHB) patients was detected by methylation-specific PCR (MSP-PCR) analysis. GSTP1mRNA expression was detected by quantitative RT-PCR. Oxidant and antioxidant levels were evaluated by plasma Malondialdehyde (MDA) adduct and GSH level detected by ELISA.
Results Thirteen of48ACLF and4of48CHB patients displayed GSTP1promoter methylated and the difference was significant (x2=5.79, P=0.03). GSTP1mRNA expression of ACLF patients was significantly decreased compared with CHB patients (P<0.01), and it was significantly decreased in methylated group of ACLF patients than unmethylated group (P=0.01). No considerable difference of clinical parameters were found between methylated and unmethylated group (P>0.05), except total bilirubin (TBIL) level (P=0.04). MDA adduct level was significantly higher in ACLF than CHB patients (12.48±4.8pmol/mg vs8.27±3.4pmol/mg, P=0.03), and GSH level was significantly lower in ACLF patients than CHB patients (0.68±0.3ng/ml versus0.42Q0.18ng/ml, P=0.02). MDA level in methylated ACLF group was higher than in unmethylated group (14.45±6.23pmol/mg versus11.76±3.94pmol/mg, P=0.04), while no significantly different GSH level was found between the two groups (0.46±0.18ng/ml versus0.41±0.18ng/ml, P=0.75). MELD was not significantly different between methylated and unmethylated group of ACLF patients (P>0.05). MDA adduct level is correlated with MELD of ACLF (r=0.579, P<0.01).
Conclusion Aberrant GSTP1promoter methylation may facilitate oxidative stress in ACLF, and oxidative stress is correlated with the severity of ACLF.
英文摘要第二部分
REDUCED GSTP1EXPRESSION IS DETECTED IN HBV-RELATED HEPATOCELLULAR CARCINOMA PATIENTS COMPARED WITH CHRONIC HEPATITIS B PATIENTS IN CHINA
Background Aberrant glutathione-S-transferase PI (GSTP1) expression and increased oxidative stress are involved in various human cancer.
Objective We aimed to assay GSTP1expression and oxidative stress status in hepatocellular carcinoma (HCC) patients and chronic hepatitis B (CHB) patients.
Methods GSTP1protein expression was detected by flow cytometry in peripheral blood mononuclear cells (PBMC) of38HCC patients,38CHB patients and10 normal contrals, while its mRNA expression level was evaluated by quantitative real-time polymerase chain reaction (QRT-PCR). Enzyme-linked-immunosorbent-assay (ELISA) was employed to measure oxidative stress status indexed with plasma levels of malondialdehyde (MDA), xanthine oxidase (XOD), and reduced glutathione hormone (GSH), glutathione-S-transferases (GST).
Results Significantly decreased GSTP1protein expression was found in HCC patients than in CHB patients. GSTP1mRNA expression in HCC patients was also decreased in contrast to CHB patients (P=0.01). Plasma MDA and XOD levels were significantly higher in HCC patients than in CHB patients, while plasma GSH and GST levels were statistically lower in HCC patients than in CHB patients.
Conclusion Reduced GSTPl expression is found in PBMC of HCC patients by flow cytometry and contributes to oxidant/antioxidant imbalance in development of HBV-related HCC from CHB in China.
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