HBV-DNA突变和细胞因子与HBV相关肝硬化和肝癌的相关性研究

发布时间：2017-12-28 06:10

本文关键词：HBV-DNA突变和细胞因子与HBV相关肝硬化和肝癌的相关性研究　出处：《南方医科大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景和目的由乙型肝炎病毒(HBV)感染引起的肝硬化及肝细胞癌仍是威胁全球人类健康的主要疾病,我国HBV感染人群达1.3亿,约15%-25%由于HBV感染相关性的疾病而死亡。对HBV高危病人早期诊断,早期发现,及时采取措施进行干预,对改善肝硬化、肝癌预后具有重要意义。目前认为HBV的基因突变及宿主体内免疫功能的改变在HBV相关的慢性肝病发生和发展中起重要作用。本研究的目的是分析HBV DNA突变以及相关的细胞因子的变化与肝硬化和肝癌的相关性,以期发现新的致病分子靶点,为将来特异性治疗HBV诱导的慢性肝病提供实验依据。研究方法1.临床实验:对象为127例慢性乙型肝炎、65例肝硬化和45例肝癌且有HBV感染证据的患者。用DNA测序法、错配扩增突变PCR法分析血清中HBV核心启动子(BCP)区T1762/A1764和前C区A1896突变情况,PCR-荧光探针法检测HBVDNA载量,用酶联免疫吸附法检测血清中细胞因子水平,用免疫组化法检测癌旁组织内细胞因子的表达定位,用realtime PCR法检测细胞或组织内分子基因的表达水平,部分指标间进行相关回归分析。2.细胞水平实验:用CCL5siRNA转染人肝癌细胞株HepG2后,用划痕实验及Transwell法观察细胞的迁移活性和能力,用Western blotting法检测蛋白表达。结果1.HBV前C区和BCP区突变与慢性肝病之间关系研究:(1)HBV的BCP区 A1762T/G1764A 和前 C 区 G1896A 的发生率(62.48%(148/237)、62.48%(148/237)和 49.79%(118/237))高于其它位点,且 A1762T 和 G1764A 是联合发生的;(2)A1762T/G1764A 和 A1762T/G1764A/G1896A 可促进 HBeAg 的转化,增加血清AFP的表达,与肝硬化和肝癌发生密切相关;(3)A1762T/G1764A突变与患者肝癌发生率呈正相关;(4)G1896A可增加HBV-DNA的复制。2.血清细胞因子和HBV相关肝硬化/肝癌的相关性研究:(1)慢乙肝病人血清中HA、LN、PC-Ⅲ和C-Ⅳ含量都非常显著的低于失代偿期肝硬化病人(P0.001);(2)CCL5、MIP-1b和HA三种因子在区分肝硬化和慢乙肝病人准确性的ROC曲线显示三种因子的AUC分别为0.8011、0.706和0.752,AUC值均大于0.7,因此,CCL5、MIP-1b和HA在区分肝硬化和慢乙肝病人中都有效,其中,CCL5准确性最高,HA次之,MIP-1b最差。3.CCL5及其受体在肝细胞肝癌中的功能研究:(1)CCL5和CCR5在肝癌组织中大量表达,明显高于肝硬化组织;(2)CCR5特异性siRNA可明显下调肝癌细胞系HepG2中CCR5转录水平;(3)特异性下调CCR5的表达后,进行划痕实验,第24小时观察到,对照组约50%的划痕被细胞迁移而修补,而CCR5siRNA转染组细胞则只迁移修补划痕的23%;(4)Transwell实验中,迁移的细胞数量在对照组为68.73±4.024,CCR5siRNA组降低至24.53±2.545(P0.001)。结论1.HBV前C区和BCP区突变,改变了病毒的复制能力,与肝硬化和肝癌发生发展密切相关。2.血清中CCL5、MIP-1b和HA水平,尤其是CCL5,可做为判断肝硬化和慢乙肝病情的指标。3.CCL5及其受体在肝癌组织中表达明显增高,在肝癌细胞的迁移和侵袭过程中起重要作用。
[Abstract]:Background and objective: cirrhosis and hepatocellular carcinoma caused by hepatitis B virus (HBV) infection is still a major threat to global health. The number of HBV infected people in China is 130 million. About 15%-25% is dead due to HBV infection related diseases. Early diagnosis of high risk patients with HBV, early detection and intervention in time are of great significance to improve the prognosis of liver cirrhosis and liver cancer. It is believed that the mutation of HBV gene and the changes in the immune function of the host play an important role in the development and development of HBV related chronic liver diseases. The purpose of this study is to analyze the correlation between HBV DNA mutation and related cytokines and liver cirrhosis and hepatocellular carcinoma, in order to discover new molecular targets, and provide experimental evidence for specific treatment of HBV induced chronic liver disease in the future. Study methods 1. clinical trials were conducted in 127 patients with chronic hepatitis B, 65 liver cirrhosis and 45 cases of liver cancer with evidence of HBV infection. Using DNA sequencing, mismatch amplification mutation PCR analysis of serum HBV core promoter (BCP) region of T1762/A1764 and C before A1896 mutation detection, HBVDNA PCR- fluorescent probe loading, the adsorption of cytokine levels in serum were detected by ELISA. The expression and localization of cytokine was detected in paracancerous tissues with immunohistochemistry, expression level was detected with PCR method in realtime cells or tissue gene, correlation and regression analysis were part of index. 2. cell level experiment: after transfection of human hepatoma cell line HepG2 with CCL5siRNA, the cell migration activity and ability were observed by scratch test and Transwell method, and the protein expression was detected by Western blotting. C and BCP mutations in 1.HBV and chronic liver disease before the relationship between the research results: (1) the incidence of HBV BCP A1762T/G1764A and former C G1896A (62.48% (148/237), 62.48% (148/237) and 49.79% (118/237)) was higher than that of other sites, and A1762T and G1764A are combined with the (2); conversion of A1762T/G1764A and A1762T/G1764A/G1896A can promote the HBeAg, increase the expression of AFP in serum, and is closely related to cirrhosis and liver cancer; (3) A1762T/G1764A mutation rate was positively correlated with the incidence of HCC patients; (4) G1896A can increase HBV-DNA replication. Study on the correlation of 2. serum cytokines and HBV related liver cirrhosis / liver cancer: (1) serum hepatitis B patients in HA, LN, slow PC- III and C- IV were very significantly lower than that of patients with decompensated liver cirrhosis (P0.001); (2) CCL5, MIP-1b and HA three factors showed that three kinds of factor AUC respectively. For the 0.8011, 0.706 and 0.752 in the ROC curve between accuracy of cirrhosis and chronic hepatitis B patients, AUC values were greater than 0.7, therefore, CCL5, MIP-1b and HA are effective in distinguishing liver cirrhosis and chronic hepatitis B patients in the highest accuracy of CCL5, HA, MIP-1b is the worst. The function of 3.CCL5 and its receptor in hepatocellular carcinoma: (1) the expression of CCL5 and CCR5 in HCC tissues was significantly higher than that of liver cirrhosis; (2) siRNA specific CCR5 could downregulate the CCR5 transcription level in hepatocellular carcinoma cell line HepG2; (3) the expression of specific downregulation of CCR5, scratch experiment twenty-fourth hours of observation, the control group was about 50% of the cell migration and scratch repair, and CCR5siRNA transfected cells only transfer repair scratches 23%; (4) in the Transwell experiment, the number of cell migration in the control group is 68.73 + 4.024, CCR5siRNA group reduced to 24.53 + 2.545 (P0.001). Conclusion the mutation of the C region and BCP region before 1.HBV changes the replication ability of the virus, which is closely related to the development of liver cirrhosis and liver cancer. 2. the level of CCL5, MIP-1b and HA in serum, especially CCL5, can be used as an indicator to judge the condition of liver cirrhosis and chronic hepatitis B. The expression of 3.CCL5 and its receptor in liver cancer tissues is significantly increased, which plays an important role in the migration and invasion of hepatoma cells.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R512.62;R575.2;R735.7

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