乙型肝炎病毒X蛋白在肝细胞癌发生发展中的作用

乙型肝炎病毒(HBV)感染是急慢性病毒性肝炎的主要致病因素,它也是诱发肝硬化,原发性肝细胞癌(HCC)的重要危险因素。据世界卫生组织(WHO)统计,每年全球范围内约有4亿人受到HBV的慢性感染,而约有1亿人死于HBV感染。乙型肝炎病毒X蛋白(HBx)是由乙型肝炎病毒X基因编码的一个由154个氨基酸组成的多功能反式激活蛋白,在病毒的复制和肿瘤的形成和发展中发挥着极其重要的作用,大量研究表明HBx与肝细胞癌的发生、发展有着密切的关系。     

人HBV、HBx定向敲入p53位点大鼠胚胎干细胞株的建立

目的 建立人HBV、HBx定向敲入p53位点的大鼠胚胎干细胞株,为建立相关动物模型奠定基础.方法 通过PCR方法扩增出人p53基因上下游同源臂、HBV全基因组序列、HBV的X蛋白编码基因(HBx),将其插入本实验室自主构建的基因打靶通用载体pKO中,分别构建pKO-gHBV及pKO-X打靶载体.载体经SalⅠ酶切线性化后,电转入状态良好的大鼠胚胎干细胞株中,经3轮药物筛选,获得单细胞克隆.通过PCR技术筛选获得阳性细胞克隆,并进行支原体污染鉴定和核型分析.结果 成功构建pKO-gHBV及pKO-X打靶载体;电转大鼠胚胎干细胞株,经3轮药物筛选,分别挑取若干细胞克隆,其中2个pKO-X细胞克隆和1个pKO-gHBV细胞克隆经PCR鉴定、支原体检测和核型分析确定结果正确.结论 成功建立了人HBV、HBx定向敲入p53位点的大鼠胚胎干细胞株,为后续建立动物模型奠定了基础.     

Replication of the hepatitis B virus requires a calcium-dependent HBx-induced G1 phase arrest of hepatocytes

Chronic HBV infections cause hepatocellular carcinoma (HCC). Activities of the HBV HBx protein regulate HBV replication and may contribute to the development of HCC. We previously reported that HBx causes primary rat hepatocytes to exit G0 but stall in G1 phase of the cell cycle; entry into G1 stimulated HBV replication. We now report that the activity of the mitochondria permeability transition pore is required for HBx regulation of cell cycle proteins and HBV replication in primary rat hepatocytes, that progression from G0 to G1 stimulates HBV polymerase activity, and that HBV replication is facilitated by the HBx-induced G1 arrest. HBx stimulation of HBV replication was linked to elevation of the R2 subunit of ribonucleotide reductase. Our studies suggest that HBx uses mitochondrial-dependent calcium signaling to cause hepatocytes to exit G0 but stall in G1 and that this HBx activity alters the cellular environment and stimulates HBV replication.     

Dissecting the pleiotropic actions of HBx mutants against hypoxia in hepatocellular carcinoma

Error-prone integration of the hepatitis B virus X protein (HBx) into the hepatocellular genome generates a multitude of mutants exerting diverse effects on the development and progression of hepatocellular carcinoma (HCC). A recent study by Lai and colleagues revealed the disparate regulatory activity of clinically-predominant HBx mutants towards hypoxia-inducible factor-1α (HIF-1α), a central regulator of tumor angiogenesis, proliferation, metastasis and differentiation. These findings have shed insight into specific viral contribution of hypoxic response during hepatocarcinogenesis.     

转基因细胞模型L02/HBx的构建及HBx对细胞周期的影响

目的:构建L02/HBx转基因细胞模型并研究HBx对肝细胞周期的影响.方法:运用脂质体转染和G418筛选获得L02/ HBx阳性克隆,并分别用RT-PCR和Western blot鉴定HBx mRNA与蛋白的表达.进一步用四唑蓝(MTT)比色试验、流式细胞仪检测L02/HBx的增殖、凋亡和细胞周期.结果:RT-PCR和Western blot分别检测到L02/ HBx细胞中HBx mRNA和蛋白的表达.MTT比色试验显示L02/HBx生长速度加快,流式细胞仪检测发现L02/HBx凋亡率低(0.09%±0.13% vs 3.74%±1.29%,P<0.05),G1期细胞比例减少(61.35%±0.82% vs 67.80±6.84%,P<0.05),S期细胞比例相应增加(36.59%±2.54% vs 22.37%±2.17%,P<0.05).经阿霉素(ADM)培养后,L02/HBx的凋亡率显著增加(34.91%±5.85% vs 0.09%±0.13%,P<0.05),G1期细胞比例明显增加但低于对照组(82.81%±6.48% vs 61.35%±0.82%,P<0.05;82.81%±6.48% vs 87.19%±1.92%,P<0.05),S期细胞比例降低但较对照组高(13.84%±6.16% vs 36.59%±2.54%,P<0.05;13.84%±6.16% vs 2.22%±1.26%,P<0.05).结论:L02/HBx构建成功,HBx能促进细胞周期进程,加快细胞的生长并抑制细胞的凋亡;转染HBx基因的肝细胞凋亡更易受凋亡因子所触发,表明HBx可能会增加正常肝细胞对诱导凋亡因素的敏感性.     

Hepatitis B virus X protein promotes proliferation and upregulates TGF-β1 and CTGF in human hepatic stellate cell line,LX-2

BACKGROUND:Chronic hepatitis B virus(HBV)infection is a major cause of liver fibrosis,but the mechanisms underlying HBV-related fibrogenesis are still unknown. Although the roles of HBV X protein(HBx)remain poorly understood,it is thought to play an important role in the regulation of cellular growth and hepatocarcinogenesis. The aim of this study was to determine the role of HBx in liver fibrogenesis by studying the effect of HBx on the proliferation and expression of fibrosis-related molecules in the huma...     

一种高效表达HBx的肝癌细胞模型的建立

目的构建稳定、高效表达HBx的转基因细胞模型BEL-7404／HBx。方法通过脂质体转染将亚克隆有HBVX基因的重组质粒pcDNA3．1（＋）-V5-HisB—HBx导人肝癌细胞系BEL-7404，并用G418筛选获取阳性克隆，分别用逆转录聚合酶链反应（RT-PCR）和免疫印迹（Westernblot）检测不同生长时期阳性克隆中HBxmRNA和蛋白的表达情况。结果不同生长时期阳性克隆均能检测到HBxmRNA和蛋白的高效表达。结论成功构建稳定、高效表达HBx的转基因肝癌细胞模型BEL-7404／HBx。     

Dicoumarol,an NQO1 inhibitor,blocks cccDNA transcription by promoting degradation of HBx

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SiRNA沉默 HBx对 HepG2.2.15细胞人端粒酶逆转录酶基因表达的影响

目的探讨应用RNA干扰技术沉默HBx对HepG2．2．15细胞中hTERT基因表达的影响。方法（1）将pSIHBV／X质粒转染HepG2．2．15细胞,RT-PCR法评估沉默效率。（2）MTT法检测转染24h、48h、72h后细胞增殖情况。（3）Real-timePCR和Westernblot检测hTERT表达情况。结果测序结果显示pSIHBV／X质粒构建正确;RT-PCR检测HBVX基因的沉默效率为53．6％;MTT检测结果显示转染24h、48h、72h后H印G2．2．15细胞的增殖受抑制,与对照组比较差异有统计学意义（P〈0．05）;Real-timePCR和Western blot检测结果均显示,转染pSIHBV／X质粒后HepG2．2．15细胞中hTERT基因的mRNA水平和蛋白水平表达分别有不同程度的下调,与对照组比较差异有统计学意义（P〈0．05）。结论siRNA沉默HBx基因可抑制HepG2．2．15细胞中癌症标志基因hTERT的表达。