【作者】 张洪涛；

【导师】 窦科峰；

【作者基本信息】 第四军医大学 ， 外科学， 2009， 博士

【摘要】 原发性肝癌(hepatocellular carcinoma, HCC)是我国最常见的恶性肿瘤之一,我国发病约占全世界发病的45%左右,而且预后不良,严重威胁着人类的生命健康。近年来,随着诊断、治疗技术的进步,多种手段的综合治疗措施已经较大地改善了肝癌患者的生存质量,但其治愈率及生存率依然不能令人满意,肝癌的复发和转移仍然是影响患者生存期的重要因素。细胞生物学及分子生物学的巨大进步为研究和探讨肝癌的起源、复发及转移提供了重要的手段,目前,我们已能够从基因水平、蛋白水平直至细胞水平等各个层次和角度来认识肝癌的起源、复发及转移,为临床治疗肝癌提供了重要的理论基础,尽管如此,我们依然缺乏对肝癌各种生物学行为更加系统的认识以及为指导临床治疗肝癌提供更加具有针对性和有效的治疗措施。因此,研究原发性肝癌的发生发展规律以指导诊断治疗一直是科研工作者们孜孜以求的目标。现在认为,原发性肝癌的发生与一些存在肝硬化的慢性肝病密切相关,是一个多因素、多阶段、多基因共同的积累过程。关于细胞癌变存在两种假说:(1)癌变是由已分化成熟的细胞去分化(退化)而引起;(2)癌变是干细胞在分化过程中受阻滞(异常分化)而引起。乙肝病毒(hepatitis B virus, HBV)的慢性感染与HCC之间存在着密切的联系,然而HBV诱发HCC的确切机制仍不十分清楚。有研究表明,乙肝病毒X基因及其编码的X蛋白(hepatitis B virus X protein, HBx)在HCC的发生中起着重要的作用。为进一步探讨HBV X基因在与HBV相关HCC发生中的作用,我们将在我科已经成功分离、纯化、培养大鼠肝干细胞的基础上,从HBV这一导致HCC发生的重要危险因素入手,以病毒HBV X基因以及其产物HBx蛋白作为对象,通过转染HBV X基因到大鼠肝干细胞,分析在肝干细胞分化过程中,HBx蛋白对其的影响,观察表达蛋白及细胞表面分子变化,进一步了解肝干细胞分化受阻的机理。为此,我们构建了带有绿色荧光蛋白的质粒,转染至大鼠肝干细胞中,通过细胞生长曲线、细胞分泌蛋白等变化,观察转染后的大鼠肝干细胞的生长情况;在裸鼠皮下注射转染HBV X基因的、转染空质粒及正常大鼠肝干细胞,观察成瘤率及肿瘤的生长情况,探讨在体内实验中HBV X基因在肝癌发生发展过程中的作用。我们首先成功的构建了质粒,为带有增强型绿色荧光蛋白报告基因HBV X基因的质粒PIRES2-EGFP-HBx。将构建好的质粒转染至大鼠肝干细胞中,我们发现携带HBx的大鼠肝干细胞尽管生长速度和大鼠肝干细胞无明显差异,但在分化过程中产生的分化细胞合成白蛋白、尿素氮的能力下降,大量表达甲胎蛋白,逐步远离肝细胞特征。转染HBV X基因、转染空质粒及正常大鼠肝干细胞在裸鼠体内成瘤能力变化的实验结果表明,转染HBV X基因组的裸鼠局部形成肿瘤,说明携带HBV X基因,表达HBx蛋白的大鼠肝干细胞在分化过程中,致瘤能力增强。以上结果提示转染HBV X基因的大鼠肝干细胞在分化过程中,受到HBV X基因的调控,不能向肝细胞或者胆管上皮细胞分化,而是增加恶性致瘤能力,向肿瘤细胞分化,初步说明了转染HBV X基因的大鼠肝干细胞可能是导致肝癌发生的始动细胞。本文初步探讨了HCC发生过程中,肝癌细胞的起源,为“原发性肝癌可能起源于肝脏干细胞的异常分化”提供更为直接的证据.,为我们提供了一个新的角度来看待肝癌的发生、发展及其转归,不仅可能为解开“肿瘤细胞的起源”这一谜团提供确凿的证据,更重要的是将为未来肿瘤防治提供新的分子靶点。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most common malignant tumors occurring in human around the world with a high mortality and conventional diagnosis and treatment of it are dismal. Although the molecular mechanisms of hepatocarcinogenesis remain poorly understood, researchers have proved that carcinogenesis of HCC involves complex biological processes, in which hepatitis B and C virus infections, aflatoxin contamination, alcohol abuse, ionizing radiation and human metabolic products toxic to the human genome are involved. Many researches have revealed that there is a strong epidemiological evidence that persistence of hepatitis B virus (HBV) infection is the most important risk factor for HCC’s development. HBV, which have the smallest genomes (approximately 3 kb) of all animal DNA viruses, specifically infect liver cells of several species, including ducks, woodchucks, squirrels, and humans. Infection of HBV usually results in acute liver damage. In some cases, however, the acute infection is not resolved and a chronic infection of the liver develops. Such chronic infection is associated with more than a hundredfold increased risk of liver cancer. HBV infection is particularly common in parts of Asia and Africa, especially in China.We know that one of the fundamental features of cancer is tumor clonality, the development of tumors from single cells that begin to proliferate abnormally. At the cellular level, the development of cancer is viewed as a multistep process involving mutation and selection for cells with progressively increasing capacity of proliferation, survival, invasion, and metastasis. The first step in the process, tumor initiation, is thought to be the result of a genetic alteration leading to abnormal proliferation of a single cell. Cell proliferation then leads to the outgrowth of a population of clonally derived tumor cells. However, the original cells leading to the development and progression of HCC remain elusive. So, one major challenge in HCC research is the identification and characterization of tumor initial cells. Some believe that cell dedifferentiation is an aberration of the normal development cycle that results in cancer, so they consider that dedifferentiated liver cells are tumor initial cells. With more knowledge about stem cells, an increasing number of scientists who have studied stem-cell treatment of cancer increasingly are examining a possible connection between stem cells and cancer in recent researches. A large number of researchers now point to liver stem cells as a possible cause of HCC because they remain alive throughout a person’s life and can receive more DNA damage than other liver cells. DNA damage causes a series of cell responses, including injury signal transduction, damage repair, and induction of cell death. The factors involved in DNA damage can also affect some aspects of the DNA injury repair system, preventing the damage from being repaired or correctly repaired, and finally leading to malignant transformations. Therefore, the accumulation of damaged DNA in liver stem cells becomes an important molecular mechanism underlying the carcinogenesis of HCC. As cell transformation by HBV is mediated by a viral gene, called the X gene that affects expression of a variety of cellular genes that drive abnormal cell proliferation and survival.Aim: To assess whether liver stem cells transfected HBV X gene differentiated differentially in vitro and to investigate whether they can cause carcinoma in nude mice.Methods: First, a plasmid containing immunoflorescent reporting protein gene with HBV X gene was constructed. Second, the plasmid was transfected into liver stem cells. The differentiation and expressions about urea nitrogen(UN)、alpha fetoprotein(AFP)、albumin(ALB) of these transfected liver stem cells were observed. Third, nude mice received subcutaneous injection of cell suspension containing these transfected liver stem cells. And the tumorigenesis rate and the growth of tumor were observed.Results: The plasmids containing the HBV X gene was successfully constructed. Levels of albumin in the differentiated cells from liver stem cells with HBV X gene were significantly lower than in normal liver stem cells, as alpha fetoprotein, higher, (p<0.05). Compared with normal liver stem cells group, nude mice received subcutaneous injection of liver stem cells with HBV X gene could develop tumor.Conclusion: Our findings demonstrate that the differentiation of liver stem cells was regulated by HBV X gene and suggest that liver stem cells with HBV X gene might be involved in hepatocarcinogenesis and could be tumor initial cells of HCC. This offers useful clue for a new approach to hepatocellular carcinoma therapy.更多还原