HBx与宿主蛋白质的相互作用

HBx是哺乳动物嗜肝性 HBV的 X读码框所编码的 HBV中唯一具有多种调控功能的病毒蛋白质。这些调控作用包括 :激活宿主细胞和病毒自身基因的转录、调控凋亡、抑制细胞中受损 DNA的外切修复反应以及激活细胞中信号转导通路如 :MAPK、JAK、STAT的级联反应等 ;HBx通过这样的多种调控作用直接或间接地对病毒自身的复制与增殖以及宿主细胞中基因的表达与调控、细胞的凋亡与癌变等过程都产生了广泛的影响     

Cooperation of tumor‐derived HBx mutants and p53‐249ser mutant in regulating cell proliferation,anchorage‐independent growth and aneuploidy in a telomerase‐immortalized normal human hepatocyte‐derived cell line

Hepatocellular carcinoma (HCC) is a common cancer, and hepatitis B virus (HBV) is a major etiological agent. Convincing epidemiological and experimental evidence also links HCC to aflatoxin, a naturally occurring mycotoxin that produces a signature p53‐249^ser mutation. Recently, we have reported that tumor‐derived HBx variants encoded by HBV exhibited attenuated transactivation and proapoptotic functions but retained their ability to block p53‐mediated apoptosis. These results indicate that mutations in HBx may contribute to the development of HCC. In this study, we determined whether tumor‐derived HBx mutants along, or in cooperation with p53‐249^ser, could alter cell proliferation and chromosome stability of normal human hepatocytes. To test this hypothesis, we established a telomerase immortalized normal human hepatocycte line HHT4 that exhibited a near diploid karyotype and expressed many hepatocyte‐specific genes. We found that overexpression one of the tumor‐derived HBx mutants, CT, significantly increased colony forming efficiency (CFE) while its corresponding wild‐type allele CNT significantly decreased CFE in HHT4 cells. p53‐249^ser rescued CNT‐mediated inhibition of colony formation. Although HHT4 cells lacked an anchorage independent growth capability as they did not form any colonies in soft agar, the CT‐expressing HHT4 cells could form colonies, which could be significantly enhanced by p53‐249^ser. Induction of aneuploidy could be observed in HHT4 cells expressing CT, but additionally recurring chromosome abnormalities could only be detected in cells coexpressing CT and p53‐249^ser. Our results are consistent with the hypothesis that certain mutations in HBx and p53 at codon 249 may cooperate in contributing to liver carcinogenesis.     

人XPD基因转染对人肝癌细胞的生物学影响

目的：XPD基因作为TFIIH因子中一员,在基因修复中发挥重大作用。本实验构建带有绿色荧光蛋白表达人野生型XPD基因重组质粒pEGFP-N2-XPD,并将其稳定转染至人肝癌细胞株Hep3B细胞,探讨XPD基因与HBx,Cdk7的相互作用,以及细胞周期和凋亡机制。方法：从人宫颈鳞癌上皮细胞株HeLa细胞克隆出人全长XPDcDNA,构建了pEGFP-N2-XPD重组体质粒,并将其稳定转染入Hep3B并筛选单克隆稳定转染重组质粒Hep3B细胞（Hep3B-pEGFP-N2/XPD）和稳定转染空载质粒Hep3B细胞（Hep3B-pEGFP-N2）。利用荧光显微镜观测绿色荧光蛋白表达,检测转染XPD基因后细胞内XPD,HBx,cdk7的表达量变化,并检测细胞的增殖凋亡以及细胞周期变化。结果：（1）利用酶切和基因测序,成功构建了带有绿色荧光蛋白表达人野生型XPD基因重组质粒pEGFP-N2-XPD。（2）免疫荧光显微镜下,Hep3B-pEGFP-N2和Hep3B-pEGFP-N2-XPD细胞中可观察到绿色荧光蛋白表达。（3）RT-PCR检测：Hep3B-pEGFP-N2/XPD细胞与Hep3B和Hep3B-pEGFP-N2,两对照组相比,HBxmRNA表达明显降低（P〈0．001）XPDmRNA表达明显增高（P〈0．01）Cdk7mRNA表达明显降低（P〈0．001）。（4）MTT检测示Hep3B-pEGFP-N2/XPD与两对照相比,细胞增殖率减弱（P〈0．05）。TUNEL检测示Hep3B-pEGFP-N2/XPD与两对照组比较,细胞凋亡明显增加（P〈0．01）。流式细胞仪检测示Hep3B-pEGFP-N2/XPD进入S期出现障碍,停滞在Go＋G1期的细胞增多,细胞凋亡率逐渐增加。（5）Westernblot检测Hep3B-pEGFP-N2/XPD细胞中XPD蛋白相对表达量均比两对照组高,Cdk7蛋白相对表达量均比两对照组低,差异有统计学意义（P〈0．001）．结论：野生型XPD基因片段成功插入pEGFP-N2空载质粒中,将其稳定转染进人肝癌细胞株Hep3B细胞中,可以在转录和蛋白水平提高细胞XPD表达、降低细胞内HBxCdk7表达。野生型XPD基因过渡表达可能抑制HBxCdk7表达来改变细胞周期、抑制细胞生长、促使细胞凋亡。     

The Effects of HBx Gene on the Expression of DNA Repair Enzymes hOGG1 and hMYHα mRNA in HepG2 Cells

To observe the alteration in the expression of DNA repair enzymes hOGG1 and hMYHa and the change in 8-OHdG levels in the HBx gene-transfected cells HepG2/HBx and to explore the mechanisms of the HBV-associated hepatocellular carcinoma, the gene-transfected cells HepG2/HBx which stably expressed HBx was established, and the effect of HBx on the cell cycle and proliferation of HepG2 was examined. By using the β-actin as the interior control, real-time polymerase chain reaction （Real-time qPCR） was employed to quantitatively detect the expression of DNA repair enzymes hOGG1 and hMYHα in the HepG2/HBx, the control cells HepG2 and HepG2 transfected with pcDNA3.1 vector （HepG2/pDNA3.1）. The 8-OHdG levels were determined by HPLC/ECD in the established gene-transfected cells HepG2/HBx and the control cells HepG2 and HepG2/pcDNA3.1. Our results showed that the expression of DNA repair enzyme hMYHα in the HepG2/HBx （0.021±0.007） was significantly lower than that of HepG2 （0.099±0.041） （P〈0.05） and HepG2/pDNA3.1 （0.121±0.005） （P〈0.05）. However, the no significant differences existed in the expression of DNA repair enzyme hOGG1 among the three cell strains （P〉0.05）. The 8-OHdG level in the HepG2/HBx was significantly higher than that in HepG2 and HepG2/pcDNA3.1 （P〈0.05）. It is concluded that HBx gene may inhibit the expression of DNA repair enzyme hMYHα mRNA to impair the ability to repair the intracellular DNA oxidative damage, to increase the oxidative DNA-adduct 8-OHdG and to affect the nucleotide excision repair function, thus participate in the occurrence and development of hepatocellular carcinoma.     

HBx、Survivin及P53在肝细胞癌中的表达及意义

目的探讨乙型肝炎病毒X蛋白（HBx）、生存素（Survivin）及抑癌基因P53在肝细胞癌（HCC）中的表达及意义。方法采用SP免疫组化方法检测58例HCC患者中HBx、Survivin及P53蛋白的表达,并分析其与临床病理特征的关系。结果 HCC组织中HBx的表达率为46.6%（27/58）。HBx蛋白阳性的HCC组织和HBx蛋白阴性的HCC组织中Survivin、P53蛋白的阳性表达率分别为70.4%（19/27）、74.1%（20/27）和35.5%（11/31）、38.7%（12/31）,组间比较差异均有统计学意义（均P〈0.05）。HBx与Survivin、P53表达呈正相关（r=0.413 6、0.432 2,P〈0.05）。HBx染色的阳性率与AFP水平和分化程度相关;Survivin染色的阳性率与TNM分期和淋巴结转移相关;P53染色的阳性率与门静脉癌栓和肿瘤直径相关。结论在HCC的发生过程中HBx蛋白可上调Survivin及P53蛋白的表达,促进HCC的发生发展     

转HBx基因肝癌细胞增殖与抗凋亡特性研究

:【目的】研究转HBx基因肝癌细胞增殖与拮抗凋亡特性 ,探讨肝癌恶性表型的分子基础。【方法】以携HBx基因重组逆转录病毒感染QGY770 1肝癌细胞 ,G418筛选 ,PCR与RT PCR鉴定 ;流式细胞仪 (FCM)检测细胞增殖周期 ;用阿霉素诱导细胞凋亡 ,FCM定量检测。【结果】QGY770 1肝癌细胞经HBx基因转染后 ,G418筛选 4～ 6周获阳性克隆株QGY/HBx ,PCR与RT -PCR分别证实细胞有HBx基因整合与HBxmRNA表达 ;细胞周期分析结果表明 ,QGY/HBx细胞的细胞周期进程明显加快 ,并可明显抵制阿霉素的凋亡诱导作用。【结论】HBx基因可加速肝癌细胞周期的进程 ,并增强肝癌细胞的抗凋亡能力。     

Low frequency of mutations in the X gene, core promoter and precore region of hepatitis B virus infected Vietnamese

Numerous mutations in the hepatitis B virus (HBV) genome have been described, but in most cases their role in the pathogenesis of HBV infection is still unclear. Therefore, we analysed specific mutations in HBV-infected Vietnamese patients and assessed their potential relationship with their clinical outcome. A total of 153 HBV-infected Vietnamese patients with well-characterised clinical profiles were enrolled. None of the study participants had a history of alcohol or drug use and none received any antiviral or immunosuppressive therapy before or during the course of this study. The HBx- and core promoter regions were analysed by sequencing. The majority of isolates corresponded to genotype A. The presence of hepatitis B e antigen (HBeAg) was associated with significantly higher viral loads in the chronic HBV-infection group (P = 0.026). Double mutations in the core promoter (1762/1764) were more frequent in those with cancer than in noncancer patients (P < 0.01). Mutations at nucleotide (nt) 1766/1773 were found at low prevalence but with no obvious association to clinical presentation. Cytosine at nt 1858 was predominant but the stop codon mutation in the precore region was not detected. In the study, 4/48 hepatocellular carcinoma (HCC) patients revealed truncated HBx, whilst the serine to alanine mutation (codon 31) of HBx was more prevalent in cancer patients than in asymptomatic HBV carriers (P < 0.01). Thus, the low frequency of mutations indicates the relation of the absence of antiviral pressure in this population. The exclusively found prevalence of certain mutations detected in those with HBV-related carcinoma nevertheless indicates a degree of association with disease progression.     

携HBx基因逆转录病毒载体的构建

【目的】构建携HBx基因的逆转录病毒载体 ,为研究HBx基因与肝癌生物学行为间的关系提供基础。【方法】采用PCR技术从HBV全基因组中扩增HBx基因 ;将HBx基因亚克隆至质粒pLNSX ,构建质粒 pLNSHBx ,磷酸钙 DNA共沉淀法将重组体导入包装细胞系PA317,检测培养上清病毒滴度。【结果】应用PCR技术成功地从HBV基因组中克隆出全长HBx基因 ,并经序列分析证实 ;建立了产病毒细胞株PA317/HBx ,检测其培养上清病毒滴度为 8.9× 10 4 CFU ,PCR证实重组病毒中含有HBx基因。【结论】成功构建了携HBx基因的较高滴度逆转录病毒载体。