HBV X基因对成人肝细胞HL-7702线粒体功能的影响

目的:研究HBV X基因与线粒体COXⅢ基因相互作用以及对线粒体功能的影响. 方法:将HBV X基因真核表达载体pCDNA3-X及空载体pCDNA3转染成人肝细胞HL-7702细胞, 经过2周G418筛选, 获得稳定表达的新细胞株, 分别命名为HL-7702/HBx和H-7702/pcDNA3, RT-PCR和Western blot方法证实HBV X基因在HL-7702/HBx细胞内的稳定表达.抽提细胞总RNA进行半定量RT-PCR及分离细胞线粒体进行Western blot检测COX Ⅲ表达水平变化, 并通过酶动力学方法检测线粒体细胞色素氧化酶活性.结果:重组质粒pcDNA3-X转染后经G418筛选, RT-PCR和Western blot分析结果显示HL-7702-HBx细胞能够稳定表达HBV X基因.RT-PCR和Western blot结果发现HBV X基因下调COX Ⅲ蛋白表达而不影响mRNA水平表达.酶动力学检测发现线粒体细胞色素C氧化酶活性降低.结论:HBV X基因通过转录后水平调节COX Ⅲ表达, HBx通过与COXⅢ相互作用抑制线粒体细胞色素氧化酶活性.     

乙肝病毒X蛋白HLA-A0201限制性CTL表位的筛选与鉴定

目的:应用T2细胞结合实验以及亲和力稳定性实验鉴定由网络表位预测服务和通用原则预测得到的乙肝病毒X蛋白(HBx)来源的HLA-A0201限制性CTL表位.方法:选择中国人群最常见的B、C基因型中的adw、adr血清型的乙肝病毒X基因序列,通过网络在线表位筛选服务,分析得到共同并且得分较高的表位,并根据超基序、延展基序及量化基序方案等通用表位筛选原则进行进一步的筛选,得到4条较为理想的九肽(HBx1,HBx2,HBx3,HBx4)作为候选表位.随后借助于流式细胞技术,通过T2细胞实验分析各候选肽以及阳性、阴性、空白对照的荧光系数,从而对所筛选的表位进行体外鉴定.结果:获得的4个候选表位 (HBx1:VLCLRPVGA,HBx2:CLFKDWEEL,HBx3:VLHKRTLGL,HBx4:HLSLRGLPV) 中HBx2的亲和力较高,HBx2和HBx4的稳定性较好.结论:CLFKDWEEL是一种乙肝病毒X蛋白潜在的HLA-A0201限制性CTL表位,下一步可通过体内试验对其免疫原性作一步的验证.     

HBx,Snail基因在肝细胞癌中的表达意义

目的:检测HBx和Snail在肝细胞癌(HCC)中表达,分析与HCC侵袭转移特性的关系.方法:西南医院全军肝胆外科研究所2006-07/2007-03手术切除肝细胞癌肿瘤组织59例,提取RNA,逆转录为eDNA.实时定量PCR(RT-PCR)定量检测HBx和Snail mRNA水平.收集、量化研究病例的肝癌临床病理特征.利用Kendall's Tau-b相关性分析,研究HBx,Snail与临床病理特征的相关性.结果:肝癌组织Snail mRNA水平与pTNM分期、门静脉侵犯呈正相关(r=0.339,P=0.029;r=0.278,P=0.039);肝癌组织HBx mRNA水平与pTNM分期、门静脉侵犯、肿瘤单发/多发、AFP水平呈正相关(r=0.330,P=0.010;r=0.289,P=0.034;r=0.340,P=0.012;r=0.299,P=0.008);肝癌组织中Snail与HBxmRNA表达呈正相关(r=0.312,P=0.006).结论:HBx,Snail与HCC侵袭转移密切相关,二者之间亦存在相关性,HBx蛋白有可能通过激活Snail转录表达提高HCC侵袭转移能力.     

Hidden secret in hepatitis B viral X protein mutation and hypoxia-inducible factor-1α in hepatocarcinoma cancer

Hepatitis B type virus (HBV) is an old hepato oncogenic and hepatitis agent. Hepatitis B viral X protein (HBx)-induced malignant transformation requires the excess amounts of ATP level, inducing the extremely oxygen-deprived condition in the cancer tissues and vessels. To adapt, cells go to shift the hypoxic responsive state by altered hypoxia-responsive molecules such as HIF-1. In addition, tumors avoid or suppress immune recognition in the energy-deprived condition. The hypoxia-inducible factor-1α (HIF-1α) regulates MAP1, histone deacetylase and MAPK pathway. In the hypoxia, the HIF-1α interacts with HIF-1β, allowing DNA binding at the hypoxia response elements (HREs), while HBx binds with the nHLH/PAS domain of HIF-1α, preventing pVHL and HIF-1α binding capacity and degradation of HIF-1α protein. Recent work of Liu et al. [2013] demonstrated that HBx in hepatocellular carcinoma (HCC) tissues contained mutations, affecting the HBx transactivation capacity and C-terminal HBx mutation. In the HCC tissues, the HBx C-terminal mutation and HIF-1α expression were related and the different C-terminal mutations of HBx exhibit the different functionality of HIF-1α. The C-terminal region of amino acids 119-140 was important for the stability and transactivation, and the point mutations K130M/V131I enhance the functionality of HIF-1α, while C-terminal truncation diminish the HIF-1α function.     

槲皮素干预下HBx基因对人肝癌细胞侵袭、迁移、增殖的影响

目的探讨槲皮素（quercetin）对人肝癌HepG2细胞及稳定转染HBX基因的HepG2细胞（HepG2-X）增殖、侵袭力的影响，并探讨其可能的机制。方法体外培养HepG2及HepG2-X细胞，分别加入10，20，40，80μmolfL槲皮素进行处理后，用MTT实验检测细胞增殖抑制率，采用涂有Matrigel胶的Transwell小室模型对传代培养的HepG2细胞进行体外侵袭力检测。结果不同浓度槲皮素均能够抑制HepG2、HepG2-X细胞的增殖，且呈时间和剂量依赖性，各HepG2-X组细胞A值均明显高于HepG2细胞（P〈O．05），HBX对HepG2细胞具有促进增殖作用。Transwell试验结果显示，与空白对照比较，槲皮素处理组HepG2细胞侵袭能力明显下降，且呈浓度依赖性。结论槲皮素能够抑制肝癌HepG2细胞的增殖、侵袭力，HBx在槲皮素的干预下能促进HepG2细胞的增殖作用。     

Evolving strategies for the treatment of hepatocellular carcinoma: From clinical-guided to molecularly-taylored therapeutic options

Hepatocellular carcinoma (hepatocellular carcinoma, HCC) is the commonest primary liver cancer (80-90%) and represents the leading cause of cancer-related death, after lung and stomach cancer.The process of neoplastic transformation proceeds through the accumulation of mutations in the genes governing cell proliferation and apoptosis.It is currently difficult to determine the natural history of patients with untreated early-stage HCC, since most with early-stage tumor patients undergoes curative therapy. Survival rates at 3 years is 65% in patients with Child-Pugh A, and single untreated lesion. This proportion increases to 70% at 5 years after radical treatment. In patients included in randomized controlled clinical trials with advanced disease, survival at 1 and 2 years is respectively 72% and 50%.Surgery is the only potentially curative treatment for HCC. In carefully selected patients, resection and transplantation in fact, allow a 5 years survival from 60% to 70%.Unfortunately most patients in Western countries present with an intermediate or advanced HCC at diagnosis with the consequent inability to use curative treatments. These patients are therefore candidates to palliative therapies that include arterial embolization and chemoembolization and systemic treatments including chemotherapy, immunotherapy and hormonal therapy. Only recently the molecular targeted drug, Sorafenib, has been introduced among the therapeutic options for these patients.     

乙型肝炎病毒X基因的原核表达及血清抗HBx抗体的检测

目的 应用表达蛋白检测乙型肝炎患者血清抗-HBx抗体水平并探讨其临床意义。方法 通过PCR扩增获得HBVX基因，与原核表达载体Pet32a＋连接构建PET32a-HBX原核表达载体，转化E．coli BL21表达获得重组融合蛋白。经切胶透析纯化后，应用重组蛋白HBx建立检测血清中抗-HBx抗体的间接ELISA方法，分别检测正常人组、急性肝炎组、慢性肝炎组、肝硬化组和肝细胞癌组患者血清中的抗-HBx抗体。结果 获得具有免疫原性的HBx融合蛋白；ELISA检测表明，慢性肝炎组、肝硬化组和肝细胞癌组的抗-HBx抗体的水平均高于急性肝炎组，差异具有显著性；在三组之问，慢性肝炎组高于肝硬化组和肝细胞癌组，差异具有显著性，肝硬化组和肝细胞癌组的抗-HBx抗体水平无显著性差异。结论 HBV患者血清中抗-HBX抗体是乙型肝炎病毒感染的一种特异性抗体，是HBV感染的血清学指标之一，可以反映乙型肝炎肝炎患者病情的变化。     

Identification of hepatitis B virus aetiologic antigens,HBx and Pre‐S2, in diffuse large B‐cell lymphoma

Diffuse large B‐cell lymphoma (DLBCL) has been reported to have a significant association with the hepatitis B virus (HBV) infection. However, there has been no experimental evidence to determine whether the components of the hepatitis B virus are expressed in lymphoid cells. In this study, we used immunohistochemical methods to explore whether the antigens of hepatitis B virus are expressed in DLBCL lymphoma cells in HBsAg‐positive DLBCL patients (HBsAg + DLBCL). HBx antigen was detected in 48.9% of HBsAg + DLBCL patients, and the expression rate of the Pre‐S2 antigen was 57.2%. HBx expression was significantly associated with high‐level expression of c‐Myc, while the Pre‐S2 antigen was not. In this study, we demonstrated that HBx antigen and Pre‐S2 antigen could be detected in lymphoma cells, and HBx expression was related to c‐Myc expression. Our findings provide a strong basis for further study of the HBV‐infected DLBCL and molecular mechanism underlying the lymphomagenesis.     

HBx mutations promote hepatoma cell migration through the Wnt/β‐catenin signaling pathway

HBx mutations (T1753V, A1762T, G1764A, and T1768A) are frequently observed in hepatitis B virus (HBV)‐related hepatocellular carcinoma (HCC). Aberrant activation of the Wnt/β‐catenin signaling pathway is involved in the development of HCC. However, activation of the Wnt/β‐catenin signaling pathway by HBx mutants has not been studied in hepatoma cells or HBV‐associated HCC samples. In this study, we examined the effects of HBx mutants on the migration and proliferation of HCC cells and evaluated the activation of Wnt/β‐catenin signaling in HBx‐transfected HCC cells and HBV‐related HCC tissues. We found that HBx mutants (T, A, TA, and Combo) promoted the migration and proliferation of hepatoma cells. The HBx Combo mutant potentiated TOP‐luc activity and increased nuclear translocation of β‐catenin. Moreover, the HBx Combo mutant increased and stabilized β‐catenin levels through inactivation of glycogen synthase kinase‐3β, resulting in upregulation of downstream target genes such as c‐Myc, CTGF, and WISP2. Enhanced activation of Wnt/β‐catenin was found in HCC tissues with HBx TA and Combo mutations. Knockdown of β‐catenin effectively abrogated cell migration and proliferation stimulated by the HBx TA and Combo mutants. Our results indicate that HBx mutants, especially the Combo mutant, allow constitutive activation of the Wnt signaling pathway and may play a pivotal role in HBV‐associated hepatocarcinogenesis.