稳定转染HBV X基因对人胆管癌细胞系RBE生物学行为的影响
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摘要
乙型肝炎病毒(Hepatitis B Virus, HBV)属于嗜肝DNA病毒科(Hepadnaviridae),感染人体后可以引起急性或暴发性肝炎、慢性肝炎,并与肝硬化(Liver Cirrhosis)以及肝细胞肝癌(Hepatocellular Carcinoma, HCC)等一系列肝脏疾病的发生发展密切相关。据估计,全球有乙肝病毒表面抗原(HBsAg)携带者3.5亿人,其中三分之一在中国,全世界每年有75万人死于HBV感染引起的疾病,其中28万人来自中国,乙肝病毒感染是一个非常严重的公共卫生问题。
HBV基因组中的X基因(HBx)及其表达产物X蛋白(HBxAg)在HBV感染相关的肝细胞癌发生发展中可能起关键作用。HBxAg自身并没有结合DNA的能力,但可与多种宿主蛋白相互作用并影响其功能,进而产生多种不同的生物学效应。细胞凋亡是为维持内环境稳定,由基因控制的细胞自主的有序死亡,涉及一系列基因的激活、表达以及调控等的作用。目前研究发现,多种肿瘤的发生并非由细胞无限增值所引起,细胞凋亡途径受阻或凋亡功能丧失已成为一些肿瘤发生的重要诱因。国内外学者此前的研究表明,HBxAg在某些实验体系中可通过抑制p53、抑制Fas信号通路,抑制caspase-3的活性等途径抑制细胞凋亡,但同时也有实验表明,HBxAg能够诱导细胞凋亡,具体机制目前仍不清楚。
近年研究发现,乙肝病毒感染可能与胆管细胞癌(Cholangiocarcinoma)的发生发展相关,是胆管癌发生的危险因子之一。例如,虽然目前尚无确切证据显示HBV可有效感染胆管细胞或在其中完成病毒生活史周期,但在某些胆管癌病例癌组织切片中可以检测到HBxAg,且其阳性率高于HBsAg,与肝癌的研究中的发现相吻合。有研究报道,HBx瞬时转染胆管癌细胞后促进癌细胞增殖,但也有实验观察到HBx转染胆管癌细胞后促进细胞凋亡
为研究乙肝病毒与胆管癌发生之间可能存在的联系,本研究分别构建了表达HBsAg、HBc/eAg和HBxAg的重组慢病毒,感染RBE胆管癌细胞后实现了相应HBV蛋白的高效率表达。进一步利用流式细胞分选技术,从上述感染细胞出发,建立了稳定表达HBsAg、HBc/eAg的RBE细胞系。
本研究还对重组慢病毒介导的HBxAg表达对RBE细胞生物学特性的影响进行了初步的分析。细胞生长曲线和克隆生长试验的结果显示,HBxAg的表达对RBE细胞的生长增殖有不利影响；而在凋亡分析试验中,未发现HBxAg的表达对RBE细胞有明显的凋亡促进效应。
通过上述工作,一方面为研究HBV蛋白对胆管细胞的效应提供了一套高效、有力的方法和工具,另一方面通过初步揭示HBxAg对RBE细胞生物学行为特性的影响,为进一步深入研究HBxAg在胆管肿瘤发生中可能起到的作用提供了线索和思路。
Hepatitis B Virus (HBV) belongs to the prototype of hepadnavirus family. HBV infection leads to acute or outbreak hepatitis, chronic hepatitis, and is closely associated with the development of liver cirrhosis and hepatocellular carcinoma (HCC). It is estimated that 350 million people are carriers of HBsAg all over the world, among which one third are Chinese, there are 750 thousand people died from disease infected with HBV, among which 280 thousand come from Chinese. HBV infection has become an extremely severe problem of public health.
HBV X gene and its expression product may play a crucial role in the development of HCC related to HBV infection. Although lacking the ability to bind to DNA, HBx protein can interact with a variety of host proteins and affect their functions which will bring about a great diversity of biological effects. In order to maintain cellular homeostasis, cells will start independent ordered dead controlled by genes, this process is called apoptosis which involved in a series of genetic activation, expression and regulation. Current studies discover that some kinds of tumors are not induced by unlimited proliferation, and that apoptotic pathway blocking or apoptotic ability losing has attributed significantly to tumorigenesis. Foreign and domestic previous study revealed that, in some experimental system, HBx protein was able to inhibit apoptosis by suppressing p53 and Fas signal passway or inactivating caspase-3. However, at the same time, other experiments showed that HBx was able to induce apoptosis, the exact mechanism is still unclear at present.
Recent study reported that HBV infection may relate to the development of cholangiocarcinoma and may be one of the risk factors of cholangiocarcinoma. For example, although exact evidence that HBV was able to infect bile duct epithelial cells or to complete viral life cycle has not acquired yet, HBx protein has been detected in some cholangiocarcinoma tissue slice, and its positive rate was higher than HBsAg. Some investigations showed that after transient transfection, HBV X gene enhanced proliferation of cholangiocarcinoma cells. Nevertheless, other experiments observed that HBV X gene promote apoptosis after transfection.
In order to study the possible relationship between HBV and the development of cholangiocarcinoma, our study constructs recombinant lentivirus expressing HBsAg, HBc/eAg and HBx protein respectively, and we also realized high-efficient expression after recombinant lentivirus infecting RBE cholangiocarcinoma cells. Based on the infected RBE sells, we further established RBE cell lines stable expressing HBsAg and HBc/eAg by utilizing fluorescence activated cell sorting.
Our study also preliminarily analysis the influence of biological property of RBE cells by expressed HBx mediated by recombinant lentivirus. The results of cellular growth curve and clone formation test revealed that HBx exerts infaust effect on the proliferation of RBE cells, however, apoptosis analysis has not detected significant proapoptotic or antiapoptotic effect yet.
Though the above-mentioned works, on the one hand, we provide a set of high-efficient methods and tools to investigate the effect of HBV protein on bile duct epithelial cells. On the other hand, we provide clues and thread to further study the possible actions of HBxAg on the development of bile duct tumor through revealing the influence of HBxAg on biological property of RBE cells.
HBV基因组中的X基因(HBx)及其表达产物X蛋白(HBxAg)在HBV感染相关的肝细胞癌发生发展中可能起关键作用。HBxAg自身并没有结合DNA的能力,但可与多种宿主蛋白相互作用并影响其功能,进而产生多种不同的生物学效应。细胞凋亡是为维持内环境稳定,由基因控制的细胞自主的有序死亡,涉及一系列基因的激活、表达以及调控等的作用。目前研究发现,多种肿瘤的发生并非由细胞无限增值所引起,细胞凋亡途径受阻或凋亡功能丧失已成为一些肿瘤发生的重要诱因。国内外学者此前的研究表明,HBxAg在某些实验体系中可通过抑制p53、抑制Fas信号通路,抑制caspase-3的活性等途径抑制细胞凋亡,但同时也有实验表明,HBxAg能够诱导细胞凋亡,具体机制目前仍不清楚。
近年研究发现,乙肝病毒感染可能与胆管细胞癌(Cholangiocarcinoma)的发生发展相关,是胆管癌发生的危险因子之一。例如,虽然目前尚无确切证据显示HBV可有效感染胆管细胞或在其中完成病毒生活史周期,但在某些胆管癌病例癌组织切片中可以检测到HBxAg,且其阳性率高于HBsAg,与肝癌的研究中的发现相吻合。有研究报道,HBx瞬时转染胆管癌细胞后促进癌细胞增殖,但也有实验观察到HBx转染胆管癌细胞后促进细胞凋亡
为研究乙肝病毒与胆管癌发生之间可能存在的联系,本研究分别构建了表达HBsAg、HBc/eAg和HBxAg的重组慢病毒,感染RBE胆管癌细胞后实现了相应HBV蛋白的高效率表达。进一步利用流式细胞分选技术,从上述感染细胞出发,建立了稳定表达HBsAg、HBc/eAg的RBE细胞系。
本研究还对重组慢病毒介导的HBxAg表达对RBE细胞生物学特性的影响进行了初步的分析。细胞生长曲线和克隆生长试验的结果显示,HBxAg的表达对RBE细胞的生长增殖有不利影响；而在凋亡分析试验中,未发现HBxAg的表达对RBE细胞有明显的凋亡促进效应。
通过上述工作,一方面为研究HBV蛋白对胆管细胞的效应提供了一套高效、有力的方法和工具,另一方面通过初步揭示HBxAg对RBE细胞生物学行为特性的影响,为进一步深入研究HBxAg在胆管肿瘤发生中可能起到的作用提供了线索和思路。
Hepatitis B Virus (HBV) belongs to the prototype of hepadnavirus family. HBV infection leads to acute or outbreak hepatitis, chronic hepatitis, and is closely associated with the development of liver cirrhosis and hepatocellular carcinoma (HCC). It is estimated that 350 million people are carriers of HBsAg all over the world, among which one third are Chinese, there are 750 thousand people died from disease infected with HBV, among which 280 thousand come from Chinese. HBV infection has become an extremely severe problem of public health.
HBV X gene and its expression product may play a crucial role in the development of HCC related to HBV infection. Although lacking the ability to bind to DNA, HBx protein can interact with a variety of host proteins and affect their functions which will bring about a great diversity of biological effects. In order to maintain cellular homeostasis, cells will start independent ordered dead controlled by genes, this process is called apoptosis which involved in a series of genetic activation, expression and regulation. Current studies discover that some kinds of tumors are not induced by unlimited proliferation, and that apoptotic pathway blocking or apoptotic ability losing has attributed significantly to tumorigenesis. Foreign and domestic previous study revealed that, in some experimental system, HBx protein was able to inhibit apoptosis by suppressing p53 and Fas signal passway or inactivating caspase-3. However, at the same time, other experiments showed that HBx was able to induce apoptosis, the exact mechanism is still unclear at present.
Recent study reported that HBV infection may relate to the development of cholangiocarcinoma and may be one of the risk factors of cholangiocarcinoma. For example, although exact evidence that HBV was able to infect bile duct epithelial cells or to complete viral life cycle has not acquired yet, HBx protein has been detected in some cholangiocarcinoma tissue slice, and its positive rate was higher than HBsAg. Some investigations showed that after transient transfection, HBV X gene enhanced proliferation of cholangiocarcinoma cells. Nevertheless, other experiments observed that HBV X gene promote apoptosis after transfection.
In order to study the possible relationship between HBV and the development of cholangiocarcinoma, our study constructs recombinant lentivirus expressing HBsAg, HBc/eAg and HBx protein respectively, and we also realized high-efficient expression after recombinant lentivirus infecting RBE cholangiocarcinoma cells. Based on the infected RBE sells, we further established RBE cell lines stable expressing HBsAg and HBc/eAg by utilizing fluorescence activated cell sorting.
Our study also preliminarily analysis the influence of biological property of RBE cells by expressed HBx mediated by recombinant lentivirus. The results of cellular growth curve and clone formation test revealed that HBx exerts infaust effect on the proliferation of RBE cells, however, apoptosis analysis has not detected significant proapoptotic or antiapoptotic effect yet.
Though the above-mentioned works, on the one hand, we provide a set of high-efficient methods and tools to investigate the effect of HBV protein on bile duct epithelial cells. On the other hand, we provide clues and thread to further study the possible actions of HBxAg on the development of bile duct tumor through revealing the influence of HBxAg on biological property of RBE cells.
引文
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