發(fā)布時(shí)間：2017-12-28 06:10

  本文關(guān)鍵詞：HBV-DNA突變和細(xì)胞因子與HBV相關(guān)肝硬化和肝癌的相關(guān)性研究　出處：《南方醫(yī)科大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 乙型肝炎病毒 基因突變 細(xì)胞因子 肝硬化 肝細(xì)胞癌 CCL5

【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細(xì)胞癌仍是威脅全球人類健康的主要疾病,我國(guó)HBV感染人群達(dá)1.3億,約15%-25%由于HBV感染相關(guān)性的疾病而死亡。對(duì)HBV高危病人早期診斷,早期發(fā)現(xiàn),及時(shí)采取措施進(jìn)行干預(yù),對(duì)改善肝硬化、肝癌預(yù)后具有重要意義。目前認(rèn)為HBV的基因突變及宿主體內(nèi)免疫功能的改變?cè)贖BV相關(guān)的慢性肝病發(fā)生和發(fā)展中起重要作用。本研究的目的是分析HBV DNA突變以及相關(guān)的細(xì)胞因子的變化與肝硬化和肝癌的相關(guān)性,以期發(fā)現(xiàn)新的致病分子靶點(diǎn),為將來(lái)特異性治療HBV誘導(dǎo)的慢性肝病提供實(shí)驗(yàn)依據(jù)。研究方法1.臨床實(shí)驗(yàn):對(duì)象為127例慢性乙型肝炎、65例肝硬化和45例肝癌且有HBV感染證據(jù)的患者。用DNA測(cè)序法、錯(cuò)配擴(kuò)增突變PCR法分析血清中HBV核心啟動(dòng)子(BCP)區(qū)T1762/A1764和前C區(qū)A1896突變情況,PCR-熒光探針法檢測(cè)HBVDNA載量,用酶聯(lián)免疫吸附法檢測(cè)血清中細(xì)胞因子水平,用免疫組化法檢測(cè)癌旁組織內(nèi)細(xì)胞因子的表達(dá)定位,用realtime PCR法檢測(cè)細(xì)胞或組織內(nèi)分子基因的表達(dá)水平,部分指標(biāo)間進(jìn)行相關(guān)回歸分析。2.細(xì)胞水平實(shí)驗(yàn):用CCL5siRNA轉(zhuǎn)染人肝癌細(xì)胞株HepG2后,用劃痕實(shí)驗(yàn)及Transwell法觀察細(xì)胞的遷移活性和能力,用Western blotting法檢測(cè)蛋白表達(dá)。結(jié)果1.HBV前C區(qū)和BCP區(qū)突變與慢性肝病之間關(guān)系研究:(1)HBV的BCP區(qū) A1762T/G1764A 和前 C 區(qū) G1896A 的發(fā)生率(62.48%(148/237)、62.48%(148/237)和 49.79%(118/237))高于其它位點(diǎn),且 A1762T 和 G1764A 是聯(lián)合發(fā)生的;(2)A1762T/G1764A 和 A1762T/G1764A/G1896A 可促進(jìn) HBeAg 的轉(zhuǎn)化,增加血清AFP的表達(dá),與肝硬化和肝癌發(fā)生密切相關(guān);(3)A1762T/G1764A突變與患者肝癌發(fā)生率呈正相關(guān);(4)G1896A可增加HBV-DNA的復(fù)制。2.血清細(xì)胞因子和HBV相關(guān)肝硬化/肝癌的相關(guān)性研究:(1)慢乙肝病人血清中HA、LN、PC-Ⅲ和C-Ⅳ含量都非常顯著的低于失代償期肝硬化病人(P0.001);(2)CCL5、MIP-1b和HA三種因子在區(qū)分肝硬化和慢乙肝病人準(zhǔn)確性的ROC曲線顯示三種因子的AUC分別為0.8011、0.706和0.752,AUC值均大于0.7,因此,CCL5、MIP-1b和HA在區(qū)分肝硬化和慢乙肝病人中都有效,其中,CCL5準(zhǔn)確性最高,HA次之,MIP-1b最差。3.CCL5及其受體在肝細(xì)胞肝癌中的功能研究:(1)CCL5和CCR5在肝癌組織中大量表達(dá),明顯高于肝硬化組織;(2)CCR5特異性siRNA可明顯下調(diào)肝癌細(xì)胞系HepG2中CCR5轉(zhuǎn)錄水平;(3)特異性下調(diào)CCR5的表達(dá)后,進(jìn)行劃痕實(shí)驗(yàn),第24小時(shí)觀察到,對(duì)照組約50%的劃痕被細(xì)胞遷移而修補(bǔ),而CCR5siRNA轉(zhuǎn)染組細(xì)胞則只遷移修補(bǔ)劃痕的23%;(4)Transwell實(shí)驗(yàn)中,遷移的細(xì)胞數(shù)量在對(duì)照組為68.73±4.024,CCR5siRNA組降低至24.53±2.545(P0.001)。結(jié)論1.HBV前C區(qū)和BCP區(qū)突變,改變了病毒的復(fù)制能力,與肝硬化和肝癌發(fā)生發(fā)展密切相關(guān)。2.血清中CCL5、MIP-1b和HA水平,尤其是CCL5,可做為判斷肝硬化和慢乙肝病情的指標(biāo)。3.CCL5及其受體在肝癌組織中表達(dá)明顯增高,在肝癌細(xì)胞的遷移和侵襲過(guò)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R512.62;R575.2;R735.7

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1 陳偉;HBV-DNA突變和細(xì)胞因子與HBV相關(guān)肝硬化和肝癌的相關(guān)性研究[D];南方醫(yī)科大學(xué);2017年

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