乙型肝炎病毒X基因在体内外对肝癌细胞增殖活性的影响

目的 探讨乙型肝炎病毒x(HBx)基因对肝癌细胞增殖活性的影响。方法将携带HBx基因的表达质粒pHA-HBx转染HepG2肝癌细胞,G418筛选阳性细胞克隆,RT-PCR鉴定HBx基因的整合及表达;通过细胞计数,观察HBx基因对肝癌细胞生长曲线和倍增时间的影响;流式细胞术检测细胞周期的变化;^3H-TdR掺入法检测细胞增殖活性;裸鼠接种观察HBx基因在体内对肝癌细胞增殖的影响。结果HBx基因在体内外对HepG2细胞的增殖活性均有明显影响,细胞生长曲线左移,倍增时间缩短;G0／G1。期细胞减少,S期和G2／M期细胞增多;转染后的细胞^3H-TdR掺入率较对照组增高;转HBx基因的裸鼠移植瘤的生长速度较对照组细胞明显加快。结论HBx基因在体内外均可提高肝癌细胞的增殖活性,增加肝癌细胞的恶性表型,加速肿瘤生长。     

乙型肝炎病毒X基因在大肠杆菌中亚克隆与鉴定

用ＨｐａⅠ和Ｂｇ１Ⅱ双酶切３２ｋｂＨＢＶＤＮＡ，得到近１１ｋｂ大小的完整ＨＢｘ基因，利用绿豆芽核酸酶将ＨＢｘ基因、载体ｐＢＲ３２２的ＢａｍＨⅠ酶切粘性末端切成平末端，在Ｔ４ＤＮＡ连接酶作用下进行体外重组后转化到大肠杆菌中，获得亚克隆Ｅ．ｃｏｌｉＪＭ１０９ｐＢＲ３２２ＨＢｘ，并经药物平板筛选、电泳分析和ＤＩＧ标记的探针做Ｓｏｕｔｈｅｒｎ印迹杂交实验证实。Ｅ．ｃｏｌｉＪＭ１０９ｐＢＲ３２２ＨＢｘ亚克隆的建立对进一步研究ＨＢｘ基因的功能、肝癌发生的机理以及乙型肝炎病毒感染的诊断等均有重要意义     

Calcium ions affect the hepatitis B virus core assembly

Previous report showed that cytosolic Ca2+ induced by hepatitis B virus X protein (HBx) promotes HBV replication. In this study, in vitro experiments showed that (i) HBV core assembly in vitro was promoted by Ca2+ through the sucrose density gradient and the analytical ultracentrifuge analysis. Also, (ii) transmission electron microscope analysis demonstrated these assembled HBV core particles were the capsids. Ex vivo experiments showed that the treatment of BAPTA-AM and cyclosporine A (CsA) reduced HBV capsids in the transfected HepG2 cells. In addition to that, the treatment of Thapsigargin (TG) increased HBV capsids in the transfected HepG2 cells. Furthermore, we investigated the increased HBV core assembly by HBx. The results show that the increased cytosolic calcium ions by HBx promote the HBV core assembly.     

Hepatocellular carcinoma: role of hepatitis B and hepatitis C viruses proteins in hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is the most important primary hepatic cancer, being a common cancer type worldwide. Many aetiological factors have been related with HCC development, such as cirrhosis, hepatitis viruses and alcohol. Chronic infection with hepatitis B (HBV) and C viruses (HCV) often results in cirrhosis and enhances the probability of developing HCC. The underlying mechanisms that lead to malignant transformation of infected cells, however, remain unclear. HBV is a DNA virus that integrates into the host genome, and this integration is believed, in part, to be carcinogenic. Besides, the virus encodes a 17 kDa protein, HBx, which is known to be a causative agent in the formation of HCC. On the contrary, HCV is a RNA virus that does not integrate into the host genome but likely induces HCC through host protein interactions or via the inflammatory response to the virus. Products encoded in the HCV genome interfere with and disturb intracellular signal transduction. Some HCV proteins, such as the core protein, NS3 and NS5A, have seen to have a regulatory effect on cellular promoters, to interact with a number of cellular proteins, and to be involved in programmed-cell death modulation under certain conditions. The identification of these proteins functions in HCC development and the subsequent development of strategies to inhibit protein-protein interactions may be the first step towards reducing the chronicity and/or of the carcinogenicity of these two viruses.     

稳定表达HBx的人肝细胞系7702的建立

目的 构建HBx表达载体,筛选可稳定表达HBx的人肝细胞(HL)-7702细胞系。 方法 采用RT-PCR法扩增HBx 基因片段,并将其连接至pIRES载体,经酶切和测序鉴定pIRES-HBx重组质粒序列。然后,分别采用Real-time PCR和Western blot技术检测验证重组质粒的正确性。最后,应用G418抗生素筛选稳定表达HBx的HL-7702细胞系。结果 经PCR扩增得到正确的HBx片段,成功构建pIRES-HBx质粒,将重组质粒转染HEK 293 细胞和HL-7702细胞均实现了HBx过量表达;经免疫荧光法鉴定,我们获得了稳定大量表达HBx的HL-7702细胞系。结论 成功构建的pIRES-HBx表达载体能在HL-7702细胞稳定表达HBx,为后续研究提供了基础实验工具。     

HBx及其截短体在乙型肝炎病毒复制中的作用

HBV感染作为引起慢性乙型肝炎、乙肝后肝硬化、原发性肝癌(hepatocellular carcinoma,HCC)的起始因素,已成为世界性的健康问题.据统计,目前世界上已有超过5亿人感染HBV,每年有1百万人死于乙肝相关疾病.HBx蛋白作为HBV的一个多功能调节蛋白,已被证实在HCC的发生过程中起了重要作用.近年来,关于乙肝病毒X蛋白(HBV X protein,HBx)影响HBV的复制研究也有了一定的进展.同时,越来越多的HBx截短体在肝病发展过程中的作用也被重视.本文将对HBx及其截短体在HBV复制中的作用作一综述.     

迷迭香酸干预下HBX基因对人肝癌细胞凋亡的影响

目的研究迷迭香酸(Rosmarinic acid)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,加入不同浓度用迷迭香酸处理后,用MTT实验检测细胞增殖抑制率,用细胞划痕和Transwell小室法测定对细胞迁移力的影响。结果不同浓度迷迭香酸均能够抑制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<0.05),显示HBX对HepG2细胞具有促进增殖作用。与对照组相比,实验组细胞划痕愈合减缓(P<0.05),Transwell穿膜细胞数明显减少(P<0.05)。且呈浓度依赖性。结论迷迭香酸能够抑制肝癌HepG2细胞的增殖、迁移及侵袭力,HBx在迷迭香酸的干预下能促进HepG2细胞凋亡的作用。