HBx蛋白调节大鼠肝干细胞分化作用的研究
|
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。
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.
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。
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.
引文
[1] Borlado LR, Mendez J. CDC6: from DNA replication to cell cycle checkpoints and oncogenesis. Carcinogenesis 2008;29 (2):237-43.
[2] Teoh NC, Dan YY, Swisshelm K, Lehman S, Wright JH, Haque J, Gu Y, Fausto N. Defective DNA strand break repair causes chromosomal instability and accelerates liver carcinogenesis in mice. Hepatology 2008;47 (6):2078-88.
[3] Hanks S, Coleman K, Reid S, Plaja A, Firth H, Fitzpatrick D, Kidd A, Mehes K, Nash R, Robin N, Shannon N, Tolmie J, Swansbury J, Irrthum A, Douglas J, Rahman N. Constitutional aneuploidy and cancer predisposition caused by biallelic mutations in BUB1B. Nat Genet 2004;36 (11):1159-61.
[4] Blasco MA. Telomerase beyond telomeres. Nat Rev Cancer 2002;2 (8):627-33.
[5] Sell S, Leffert HL. Liver cancer stem cells. J Clin Oncol 2008;26 (17):2800-5.
[6] Bachoo RM, Maher EA, Ligon KL, Sharpless NE, Chan SS, You MJ, Tang Y, DeFrances J, Stover E, Weissleder R, Rowitch DH, Louis DN, DePinho RA. Epidermal growth factor receptor and Ink4a/Arf: convergent mechanisms governing terminal differentiation and transformation along the neural stem cell to astrocyte axis. Cancer Cell 2002;1 (3):269-77.
[7] Beachy PA, Karhadkar SS, Berman DM. Tissue repair and stem cell renewal in carcinogenesis. Nature 2004;432 (7015):324-31.
[8] Calabrese P, Tavare S, Shibata D. Pretumor progression: clonal evolution of human stem cell populations. Am J Pathol 2004;164 (4):1337-46.
[9] Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature 2001;414 (6859):105-11.
[10] Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997;3 (7):730-7.
[11] George AA, Franklin J, Kerkof K, Shah AJ, Price M, Tsark E, Bockstoce D, Yao D, Hart N, Carcich S, Parkman R, Crooks GM, Weinberg K. Detection of leukemic cells in the CD34(+)CD38(-) bone marrow progenitor population in children with acute lymphoblastic leukemia. Blood 2001;97 (12):3925-30.
[12] Mauro MJ, Druker BJ. Chronic myelogenous leukemia. Curr Opin Oncol 2001;13 (1):3-7.
[13] Heim D, Wege H. Hepatic stem and progenitor cells in liver diseases and hepatocarcinogenesis. Minerva Gastroenterol Dietol 2009;55 (2):111-21.
[14] Houghton J, Stoicov C, Nomura S, Rogers AB, Carlson J, Li H, Cai X, Fox JG, Goldenring JR, Wang TC. Gastric cancer originating from bone marrow-derived cells. Science 2004;306 (5701):1568-71.
[15] Kim CF, Jackson EL, Woolfenden AE, Lawrence S, Babar I, Vogel S, Crowley D, Bronson RT, Jacks T. Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 2005;121 (6):823-35.
[16] Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 2003;100 (7):3983-8.
[17]Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, Dirks PB. Identification of a cancer stem cell in human brain tumors. Cancer Res 2003;63 (18):5821-8.
[18] Jordan CT. Cancer stem cell biology: from leukemia to solid tumors. Curr Opin Cell Biol 2004;16 (6):708-12.
[19] Schalken JA, van Leenders G. Cellular and molecular biology of the prostate: stem cell biology. Urology 2003;62 (5 Suppl 1):11-20.
[20] Pour PM, Schmied B. The link between exocrine pancreatic cancer and the endocrine pancreas. Int J Pancreatol 1999;25 (2):77-87.
[21] Polyak K, Hahn WC. Roots and stems: stem cells in cancer. Nat Med 2006;12 (3):296-300.
[22] Kai T, Spradling A. Differentiating germ cells can revert into functional stem cells in Drosophila melanogaster ovaries. Nature 2004;428 (6982):564-9.
[23] Grozdanov PN, Yovchev MI, Dabeva MD. The oncofetal protein glypican-3 is a novel marker of hepatic progenitor/oval cells. Lab Invest 2006;86 (12):1272-84.
[24] Sell S. Cellular origin of hepatocellular carcinomas. Semin Cell Dev Biol 2002;13 (6):419-24.
[25] Mitaka T. Reconstruction of hepatic organoid by hepatic stem cells. J Hepatobiliary Pancreat Surg 2002;9 (6):697-703.
[26] Chen Q, Kon J, Ooe H, Sasaki K, Mitaka T. Selective proliferation of rat hepatocyte progenitor cells in serum-free culture. Nat Protoc 2007;2 (5):1197-205.
[27] Gordon GJ, Coleman WB, Grisham JW. Temporal analysis of hepatocyte differentiation by small hepatocyte-like progenitor cells during liver regeneration in retrorsine-exposed rats. Am J Pathol 2000;157 (3):771-86.
[28] Gordon GJ, Coleman WB, Hixson DC, Grisham JW. Liver regeneration in rats with retrorsine-induced hepatocellular injury proceeds through a novel cellular response. Am J Pathol 2000;156 (2):607-19.
[29] Malhi H, Irani AN, Gagandeep S, Gupta S. Isolation of human progenitor liver epithelial cells with extensive replication capacity and differentiation into mature hepatocytes. J Cell Sci 2002;115 (Pt 13):2679-88.
[30]苏娟,姚玉成,王忠华,王新民,马大烈,余宏宇,熊俊,訾晓渊,赵书民,胡以平.干细胞样原始肝细胞的分离和鉴定.癌变畸变突变2000;12(4):200-201.
[31] Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, Boggs SS, Greenberger JS, Goff JP. Bone marrow as a potential source of hepatic oval cells. Science 1999;284 (5417):1168-70.
[32]Avital I, Inderbitzin D, Aoki T, Tyan DB, Cohen AH, Ferraresso C, Rozga J, Arnaout WS, Demetriou AA. Isolation, characterization, and transplantation of bone marrow-derived hepatocyte stem cells. Biochem Biophys Res Commun 2001;288 (1):156-64.
[33] Avital I, Feraresso C, Aoki T, Hui T, Rozga J, Demetriou A, Muraca M. Bone marrow-derived liver stem cell and mature hepatocyte engraftment in livers undergoing rejection. Surgery 2002;132 (2):384-90.
[34] Inderbitzin D, Avital I, Gloor B, Keogh A, Candinas D. Functional comparison of bone marrow-derived liver stem cells: selection strategy for cell-based therapy. J Gastrointest Surg 2005;9 (9):1340-5.
[35] Harris JR, Brown GA, Jorgensen M, Kaushal S, Ellis EA, Grant MB, Scott EW. Bone marrow-derived cells home to and regenerate retinal pigment epithelium after injury. Invest Ophthalmol Vis Sci 2006;47 (5):2108-13.
[36] Inderbitzin D, Avital I, Keogh A, Beldi G, Quarta M, Gloor B, Candinas D. Interleukin-3 induces hepatocyte-specific metabolic activity in bone marrow-derived liver stem cells. J Gastrointest Surg 2005;9 (1):69-74.
[37] Dabeva MD, Hwang SG, Vasa SR, Hurston E, Novikoff PM, Hixson DC, Gupta S, Shafritz DA. Differentiation of pancreatic epithelial progenitor cells into hepatocytes following transplantation into rat liver. Proc Natl Acad Sci U S A 1997;94 (14):7356-61.
[38] Shen CN, Slack JM, Tosh D. Molecular basis of transdifferentiation of pancreas to liver. Nat Cell Biol 2000;2 (12):879-87.
[39] Hay DC, Zhao D, Ross A, Mandalam R, Lebkowski J, Cui W. Direct differentiation of human embryonic stem cells to hepatocyte-like cells exhibiting functional activities. Cloning Stem Cells 2007;9 (1):51-62.
[40] Weber A. Immortalization of hepatic progenitor cells. Pathol Biol (Paris) 2004;52 (2):93-6.
[41] Kamiya A, Gonzalez FJ, Nakauchi H. Identification and differentiation of hepatic stem cells during liver development. Front Biosci 2006;11:1302-10.
[42] Grisham JW, Coleman WB, Smith GJ. Isolation, culture, and transplantation of rat hepatocytic precursor (stem-like) cells. Proc Soc Exp Biol Med 1993;204 (3):270-9.
[43] Nagai H, Terada K, Watanabe G, Ueno Y, Aiba N, Shibuya T, Kawagoe M, Kameda T, Sato M, Senoo H, Sugiyama T. Differentiation of liver epithelial (stem-like) cells into hepatocytes induced by coculture with hepatic stellate cells. Biochem Biophys Res Commun 2002;293 (5):1420-5.
[44] Germain L, Noel M, Gourdeau H, Marceau N. Promotion of growth and differentiation of rat ductular oval cells in primary culture. Cancer Res 1988;48 (2):368-78.
[45] Lazaro CA, Rhim JA, Yamada Y, Fausto N. Generation of hepatocytes from oval cell precursors in culture. Cancer Res 1998;58 (23):5514-22.
[46] Jung Y, Oh SH, Zheng D, Shupe TD, Witek RP, Petersen BE. A potential role of somatostatin and its receptor SSTR4 in the migration of hepatic oval cells. Lab Invest 2006;86 (5):477-89.
[47] Yang L, Li S, Hatch H, Ahrens K, Cornelius JG, Petersen BE, Peck AB. In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells. Proc Natl Acad Sci U S A 2002;99 (12):8078-83.
[48] Oertel M, Menthena A, Dabeva MD, Shafritz DA. Cell competition leads to a high level of normal liver reconstitution by transplanted fetal liver stem/progenitor cells. Gastroenterology 2006;130 (2):507-20; quiz 90.
[49] Lemmer ER, Vessey CJ, Gelderblom WC, Shephard EG, Van Schalkwyk DJ, Van Wijk RA, Marasas WF, Kirsch RE, Hall Pde L. Fumonisin B1-induced hepatocellular and cholangiocellular tumors in male Fischer 344 rats: potentiating effects of 2-acetylaminofluorene on oval cell proliferation and neoplastic development in a discontinued feeding study. Carcinogenesis 2004;25 (7):1257-64.
[50] Sharma AD, Cantz T, Manns MP, Ott M. The role of stem cells in physiology, pathophysiology, and therapy of the liver. Stem Cell Rev 2006;2 (1):51-8.
[51] Baumann U, Crosby HA, Ramani P, Kelly DA, Strain AJ. Expression of the stem cell factor receptor c-kit in normal and diseased pediatric liver: identification of a human hepatic progenitor cell? Hepatology 1999;30 (1):112-7.
[52] Dumble ML, Croager EJ, Yeoh GC, Quail EA. Generation and characterization of p53 null transformed hepatic progenitor cells: oval cells give rise to hepatocellular carcinoma. Carcinogenesis 2002;23 (3):435-45.
[53] Dumble ML, Knight B, Quail EA, Yeoh GC. Hepatoblast-like cells populate the adult p53 knockout mouse liver: evidence for a hyperproliferative maturation-arrested stem cell compartment. Cell Growth Differ 2001;12 (5):223-31.
[54] Lowes KN, Brennan BA, Yeoh GC, Olynyk JK. Oval cell numbers in human chronic liver diseases are directly related to disease severity. Am J Pathol 1999;154 (2):537-41.
[55] Sell S, Salman J. Light- and electron-microscopic autoradiographic analysis of proliferating cells during the early stages of chemical hepatocarcinogenesis in the rat induced by feeding N-2-fluorenylacetamide in a choline-deficient diet. Am J Pathol 1984;114 (2):287-300.
[56] Li YG, Chen M, Zhang DZ, Wang ZY, Zeng WQ, Shi XF, Guo Y, Guo SH, Ren H. [Clinical research on the treatment effect of autologous dendritic cell vaccine on the patients with chronic hepatitis B]. Zhonghua Gan Zang Bing Za Zhi 2003;11 (4):206-8.
[57] Chen M, Li YG, Zhang DZ, Wang ZY, Zeng WQ, Shi XF, Guo Y, Guo SH, Ren H. Therapeutic effect of autologous dendritic cell vaccine on patients with chronic hepatitis B: a clinical study. World J Gastroenterol 2005;11 (12):1806-8.
[58] Cai DC, Li J, Zeng Y, Li YG, Ren H. [A study on the anti-HBV effect of dendritic cell from human umbilical cord blood]. Zhonghua Gan Zang Bing Za Zhi 2007;15 (2):88-91.
[59] Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55 (2):74-108.
[60] Bergsland EK. Molecular mechanisms underlying the development of hepatocellular carcinoma. Semin Oncol 2001;28 (5):521-31.
[61] Thorgeirsson SS, Grisham JW. Molecular pathogenesis of human hepatocellular carcinoma. Nat Genet 2002;31 (4):339-46.
[62] Satyanarayana A, Manns MP, Rudolph KL. Telomeres and telomerase: a dual role in hepatocarcinogenesis. Hepatology 2004;40 (2):276-83.
[63] Brechot C. Pathogenesis of hepatitis B virus-related hepatocellular carcinoma: old and new paradigms. Gastroenterology 2004;127 (5 Suppl 1):S56-61.
[64] Velazquez RF, Rodriguez M, Navascues CA, Linares A, Perez R, Sotorrios NG, Martinez I, Rodrigo L. Prospective analysis of risk factors for hepatocellular carcinoma in patients with liver cirrhosis. Hepatology 2003;37 (3):520-7.
[65] Hassan MM, Hwang LY, Hatten CJ, Swaim M, Li D, Abbruzzese JL, Beasley P, Patt YZ. Risk factors for hepatocellular carcinoma: synergism of alcohol with viral hepatitis and diabetes mellitus. Hepatology 2002;36 (5):1206-13.
[66] Ming L, Thorgeirsson SS, Gail MH, Lu P, Harris CC, Wang N, Shao Y, Wu Z, Liu G, Wang X, Sun Z. Dominant role of hepatitis B virus and cofactor role of aflatoxin in hepatocarcinogenesis in Qidong, China. Hepatology 2002;36 (5):1214-20.
[67] Ohata K, Hamasaki K, Toriyama K, Matsumoto K, Saeki A, Yanagi K, Abiru S, Nakagawa Y, Shigeno M, Miyazoe S, Ichikawa T, Ishikawa H, Nakao K, Eguchi K. Hepatic steatosis is a risk factor for hepatocellular carcinoma in patients with chronic hepatitis C virus infection. Cancer 2003;97 (12):3036-43.
[68] Yano Y, Yamashita F, Sumie S, Ando E, Fukumori K, Kiyama M, Oyama T, Kuroki S, Kato O, Yamamoto H, Tanaka M, Sata M. Clinical features of hepatocellular carcinoma seronegative for both HBsAg and anti-HCV antibody but positive for anti-HBc antibody in Japan. Am J Gastroenterol 2002;97 (1):156-61.
[69] Pollicino T, Squadrito G, Cerenzia G, Cacciola I, Raffa G, Craxi A, Farinati F, Missale G, Smedile A, Tiribelli C, Villa E, Raimondo G. Hepatitis B virus maintains its pro-oncogenic properties in the case of occult HBV infection. Gastroenterology 2004;126 (1):102-10.
[70] Lok AS, McMahon BJ. Chronic hepatitis B: update of recommendations. Hepatology 2004;39 (3):857-61.
[71] Van Damme P, Van Herck K. A review of the efficacy, immunogenicity and tolerability of a combined hepatitis A and B vaccine. Expert Rev Vaccines 2004;3 (3):249-67.
[72]梁晓峰,陈园生,王晓军,贺雄,陈丽娟,王骏,林长缨,白呼群,严俊,崔钢,于竞进.中国3岁以上人群乙型肝炎血清流行病学研究.中华流行病学杂志2005,26(9):655-658.
[73]中华医学会第十二次全国病毒性肝炎及肝病学术会议纪要.中华传染病杂志2005,25(4): 286-287.
[74] Paterlini-Brechot P, Saigo K, Murakami Y, Chami M, Gozuacik D, Mugnier C, Lagorce D, Brechot C. Hepatitis B virus-related insertional mutagenesis occurs frequently in human liver cancers and recurrently targets human telomerase gene. Oncogene 2003;22 (25):3911-6.
[75] Murakami Y, Saigo K, Takashima H, Minami M, Okanoue T, Brechot C, Paterlini-Brechot P. Large scaled analysis of hepatitis B virus (HBV) DNA integration in HBV related hepatocellular carcinomas. Gut 2005;54(8):1162-8.
[76] Toyoda H, Kumada T, Kaneoka Y, Murakami Y. Impact of hepatitis B virus (HBV) X gene integration in liver tissue on hepatocellular carcinoma development in serologically HBV-negative chronic hepatitis C patients. J Hepatol 2008;48 (1):43-50.
[77] Herath NI, Leggett BA, MacDonald GA. Review of genetic and epigenetic alterations in hepatocarcinogenesis. J Gastroenterol Hepatol 2006;21 (1 Pt 1):15-21.
[78] Cougot D, Neuveut C, Buendia MA. HBV induced carcinogenesis. J Clin Virol 2005;34 Suppl 1:S75-8.
[79] Cui F, Wang Y, Wang J, Wei K, Hu J, Liu F, Wang H, Zhao X, Zhang X, Yang X. The up-regulation of proteasome subunits and lysosomal proteases in hepatocellular carcinomas of the HBx gene knockin transgenic mice. Proteomics 2006;6 (2):498-504.
[80] Wu CG, Forgues M, Siddique S, Farnsworth J, Valerie K, Wang XW. SAGE transcript profiles of normal primary human hepatocytes expressing oncogenic hepatitis B virus X protein. Faseb J 2002;16 (12):1665-7.
[81] Block TM, Mehta AS, Fimmel CJ, Jordan R. Molecular viral oncology of hepatocellular carcinoma. Oncogene 2003;22 (33):5093-107.
[82] Dewantoro O, Gani RA, Akbar N. Hepatocarcinogenesis in viral Hepatitis B infection: the role of HBx and p53. Acta Med Indones 2006;38 (3):154-9.
[83] Ahn JY, Chung EY, Kwun HJ, Jang KL. Transcriptional repression of p21(waf1) promoter by hepatitis B virus X protein via a p53-independent pathway. Gene 2001;275 (1):163-8.
[84] Tang H, Da L, Mao Y, Li Y, Li D, Xu Z, Li F, Wang Y, Tiollais P, Li T, Zhao M. Hepatitis B virus X protein sensitizes cells to starvation-inducedautophagy via up-regulation of beclin 1 expression. Hepatology 2009;49 (1):60-71.
[85] Zhang H, Shan CL, Li N, Zhang X, Zhang XZ, Xu FQ, Zhang S, Qiu LY, Ye LH, Zhang XD. Identification of a natural mutant of HBV X protein truncated 27 amino acids at the COOH terminal and its effect on liver cell proliferation. Acta Pharmacol Sin 2008;29 (4):473-80.
[86] Zhang X, Dong N, Yin L, Cai N, Ma H, You J, Zhang H, Wang H, He R, Ye L. Hepatitis B virus X protein upregulates survivin expression in hepatoma tissues. J Med Virol 2005;77 (3):374-81.
[87] Zhang X, Dong N, Zhang H, You J, Wang H, Ye L. Effects of hepatitis B virus X protein on human telomerase reverse transcriptase expression and activity in hepatoma cells. J Lab Clin Med 2005;145 (2):98-104.
[88] Lee YI, Han YJ, Lee SY, Lee YI, Park SK, Park YJ, Moon HB, Shin JH, Lee JH. Activation of insulin-like growth factor II signaling by mutant type p53: physiological implications for potentiation of IGF-II signaling by p53 mutant 249. Mol Cell Endocrinol 2003;203 (1-2):51-63.
[89] Fan ZR, Yang DH, Cui J, Qin HR, Huang CC. Expression of insulin like growth factor II and its receptor in hepatocellular carcinogenesis. World J Gastroenterol 2001;7 (2):285-8.
[90] Alexia C, Fourmatgeat P, Delautier D, Groyer A. Insulin-like growth factor-I stimulates H4II rat hepatoma cell proliferation: dominant role of PI-3'K/Akt signaling. Exp Cell Res 2006;312 (7):1142-52.
[91] Wei W, Huang W, Pan Y, Zhu F, Wu J. Functional switch of viral protein HBx on cell apoptosis, transformation, and tumorigenesis in association with oncoprotein Ras. Cancer Lett 2006;244 (1):119-28.
[92] Huang J, Wu K, Zhang J, Si W, Zhu Y, Wu J. Putative tumor suppressorYueF affects the functions of hepatitis B virus X protein in hepatoma cell apoptosis and p53 expression. Biotechnol Lett 2008;30 (2):235-42.
[93] Shih WL, Kuo ML, Chuang SE, Cheng AL, Doong SL. Hepatitis B virus X protein inhibits transforming growth factor-beta -induced apoptosis through the activation of phosphatidylinositol 3-kinase pathway. J Biol Chem 2000;275 (33):25858-64.
[94] Yoo YG, Lee MO. Hepatitis B virus X protein induces expression of Fas ligand gene through enhancing transcriptional activity of early growth response factor. J Biol Chem 2004;279 (35):36242-9.
[95] Yi YS, Park SG, Byeon SM, Kwon YG, Jung G. Hepatitis B virus X protein induces TNF-alpha expression via down-regulation of selenoprotein P in human hepatoma cell line, HepG2. Biochim Biophys Acta 2003;1638 (3):249-56.
[96] Kim KH, Seong BL. Pro-apoptotic function of HBV X protein is mediated by interaction with c-FLIP and enhancement of death-inducing signal. Embo J 2003;22 (9):2104-16.
[97] Chami M, Ferrari D, Nicotera P, Paterlini-Brechot P, Rizzuto R. Caspase-dependent alterations of Ca2+ signaling in the induction of apoptosis by hepatitis B virus X protein. J Biol Chem 2003;278 (34):31745-55.
[98] Chisari FV, Pinkert CA, Milich DR, Filippi P, McLachlan A, Palmiter RD, Brinster RL. A transgenic mouse model of the chronic hepatitis B surface antigen carrier state. Science 1985;230 (4730):1157-60.
[99] Xiong J, Yao YC, Zi XY, Li JX, Wang XM, Ye XT, Zhao SM, Yan YB, Yu HY, Hu YP. Expression of hepatitis B virus X protein in transgenic mice. World J Gastroenterol 2003;9 (1):112-6.
[100]Gripon P, Diot C, Theze N, Fourel I, Loreal O, Brechot C, Guguen-Guillouzo C. Hepatitis B virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide. J Virol 1988;62 (11):4136-43.
[101]Diot C, Gripon P, Rissel M, Guguen-Guillouzo C. Replication of hepatitis B virus in differentiated adult rat hepatocytes transfected with cloned viral DNA. J Med Virol 1992;36 (2):93-100.
[102]Wu CH, Ouyang EC, Walton C, Promrat K, Forouhar F, Wu GY. Hepatitis B virus infection of transplanted human hepatocytes causes a biochemical and histological hepatitis in immunocopetentent rats. World J Gastroenterol 2003;9 (5):978-83.
[103]蒋黎,毛青,王宇明,李俊刚,刘俊. HBV感染的人鼠嵌合肝动物模型的建立.第三军医大学学报2003,25(5):120-122.
[104]谭龙益,叶廷军,王皓,孔宪涛;乙型肝炎病毒基因注入大鼠肝脏后的短暂抗原表达.第二军医大学学报1998,16(2):73-75.
[105]姚云清,张定凤,罗云,张大志,黄爱龙,王波,周卫平,任红,郭树华.乙型肝炎病毒裸DNA转染原代大鼠肝细胞模型的建立.中华肝脏病杂志2002,10(4); 36-40.
[106]于恒超李韧千年松窦科峰.大鼠肝硬化模型中小肝细胞分离培养方法的建立及其表型分析细胞与分子免疫学杂志2007;23(12):1203-1204.
[107]Zheng Y, Chen WL, Louie SG, Yen TS, Ou JH. Hepatitis B virus promotes hepatocarcinogenesis in transgenic mice. Hepatology 2007;45 (1):16-21.
[108]Zhu H, Wang Y, Chen J, Cheng G, Xue J. Transgenic mice expressing hepatitis B virus X protein are more susceptible to carcinogen induced hepatocarcinogenesis. Exp Mol Pathol 2004;76 (1):44-50.
[109]Slagle BL, Lee TH, Medina D, Finegold MJ, Butel JS. Increasedsensitivity to the hepatocarcinogen diethylnitrosamine in transgenic mice carrying the hepatitis B virus X gene. Mol Carcinog 1996;15 (4):261-9.
[110]Reid LM, Jefferson DM. Culturing hepatocytes and other differentiated cells. Hepatology 1984;4 (3):548-59.
[111]Runge D, Runge DM, Jager D, Lubecki KA, Beer Stolz D, Karathanasis S, Kietzmann T, Strom SC, Jungermann K, Fleig WE, Michalopoulos GK. Serum-free, long-term cultures of human hepatocytes: maintenance of cell morphology, transcription factors, and liver-specific functions. Biochem Biophys Res Commun 2000;269 (1):46-53.
[112]Fromm ME, Taylor LP, Walbot V. Stable transformation of maize after gene transfer by electroporation. Nature 1986;319 (6056):791-3.
[113] Nakamura T, Nawa K, Ichihara A. Partial purification and characterization of hepatocyte growth factor from serum of hepatectomized rats. Biochem Biophys Res Commun 1984;122 (3):1450-9.
[114]Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Largaespada DA, Verfaillie CM. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418 (6893):41-9.
[115]Shirakata Y, Kawada M, Fujiki Y, Sano H, Oda M, Yaginuma K, Kobayashi M, Koike K. The X gene of hepatitis B virus induced growth stimulation and tumorigenic transformation of mouse NIH3T3 cells. Jpn J Cancer Res 1989;80 (7):617-21.
[116]Luber B, Arnold N, Sturzl M, Hohne M, Schirmacher P, Lauer U, Wienberg J, Hofschneider PH, Kekule AS. Hepatoma-derived integrated HBV DNA causes multi-stage transformation in vitro. Oncogene 1996;12(8):1597-608.
[117]李涛,刘国珍,谭德明,吴传湘,刘洪波.乙肝病毒x基因真核表达质粒及细胞模型的建立.生命科学研究2007;11(4):355-360.
[118]Sell S, Xu KL, Huff WE, Kabena LF, Harvery RB, Dunsford HA. Aflatoxin exposure produces serum alphafetoprotein elevations and marked oval cell proliferation in young male Pekin ducklings. Pathology 1998;30 (1):34-9.
[119]Coleman WB, Wennerberg AE, Smith GJ, Grisham JW. Regulation of the differentiation of diploid and some aneuploid rat liver epithelial (stemlike) cells by the hepatic microenvironment. Am J Pathol 1993;142 (5):1373-82.
[120]Steinberg P, Steinbrecher R, Radaeva S, Schirmacher P, Dienes HP, Oesch F, Bannasch P. Oval cell lines OC/CDE 6 and OC/CDE 22 give rise to cholangio-cellular and undifferentiated carcinomas after transformation. Lab Invest 1994;71 (5):700-9.
[2] Teoh NC, Dan YY, Swisshelm K, Lehman S, Wright JH, Haque J, Gu Y, Fausto N. Defective DNA strand break repair causes chromosomal instability and accelerates liver carcinogenesis in mice. Hepatology 2008;47 (6):2078-88.
[3] Hanks S, Coleman K, Reid S, Plaja A, Firth H, Fitzpatrick D, Kidd A, Mehes K, Nash R, Robin N, Shannon N, Tolmie J, Swansbury J, Irrthum A, Douglas J, Rahman N. Constitutional aneuploidy and cancer predisposition caused by biallelic mutations in BUB1B. Nat Genet 2004;36 (11):1159-61.
[4] Blasco MA. Telomerase beyond telomeres. Nat Rev Cancer 2002;2 (8):627-33.
[5] Sell S, Leffert HL. Liver cancer stem cells. J Clin Oncol 2008;26 (17):2800-5.
[6] Bachoo RM, Maher EA, Ligon KL, Sharpless NE, Chan SS, You MJ, Tang Y, DeFrances J, Stover E, Weissleder R, Rowitch DH, Louis DN, DePinho RA. Epidermal growth factor receptor and Ink4a/Arf: convergent mechanisms governing terminal differentiation and transformation along the neural stem cell to astrocyte axis. Cancer Cell 2002;1 (3):269-77.
[7] Beachy PA, Karhadkar SS, Berman DM. Tissue repair and stem cell renewal in carcinogenesis. Nature 2004;432 (7015):324-31.
[8] Calabrese P, Tavare S, Shibata D. Pretumor progression: clonal evolution of human stem cell populations. Am J Pathol 2004;164 (4):1337-46.
[9] Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature 2001;414 (6859):105-11.
[10] Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997;3 (7):730-7.
[11] George AA, Franklin J, Kerkof K, Shah AJ, Price M, Tsark E, Bockstoce D, Yao D, Hart N, Carcich S, Parkman R, Crooks GM, Weinberg K. Detection of leukemic cells in the CD34(+)CD38(-) bone marrow progenitor population in children with acute lymphoblastic leukemia. Blood 2001;97 (12):3925-30.
[12] Mauro MJ, Druker BJ. Chronic myelogenous leukemia. Curr Opin Oncol 2001;13 (1):3-7.
[13] Heim D, Wege H. Hepatic stem and progenitor cells in liver diseases and hepatocarcinogenesis. Minerva Gastroenterol Dietol 2009;55 (2):111-21.
[14] Houghton J, Stoicov C, Nomura S, Rogers AB, Carlson J, Li H, Cai X, Fox JG, Goldenring JR, Wang TC. Gastric cancer originating from bone marrow-derived cells. Science 2004;306 (5701):1568-71.
[15] Kim CF, Jackson EL, Woolfenden AE, Lawrence S, Babar I, Vogel S, Crowley D, Bronson RT, Jacks T. Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 2005;121 (6):823-35.
[16] Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A 2003;100 (7):3983-8.
[17]Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, Dirks PB. Identification of a cancer stem cell in human brain tumors. Cancer Res 2003;63 (18):5821-8.
[18] Jordan CT. Cancer stem cell biology: from leukemia to solid tumors. Curr Opin Cell Biol 2004;16 (6):708-12.
[19] Schalken JA, van Leenders G. Cellular and molecular biology of the prostate: stem cell biology. Urology 2003;62 (5 Suppl 1):11-20.
[20] Pour PM, Schmied B. The link between exocrine pancreatic cancer and the endocrine pancreas. Int J Pancreatol 1999;25 (2):77-87.
[21] Polyak K, Hahn WC. Roots and stems: stem cells in cancer. Nat Med 2006;12 (3):296-300.
[22] Kai T, Spradling A. Differentiating germ cells can revert into functional stem cells in Drosophila melanogaster ovaries. Nature 2004;428 (6982):564-9.
[23] Grozdanov PN, Yovchev MI, Dabeva MD. The oncofetal protein glypican-3 is a novel marker of hepatic progenitor/oval cells. Lab Invest 2006;86 (12):1272-84.
[24] Sell S. Cellular origin of hepatocellular carcinomas. Semin Cell Dev Biol 2002;13 (6):419-24.
[25] Mitaka T. Reconstruction of hepatic organoid by hepatic stem cells. J Hepatobiliary Pancreat Surg 2002;9 (6):697-703.
[26] Chen Q, Kon J, Ooe H, Sasaki K, Mitaka T. Selective proliferation of rat hepatocyte progenitor cells in serum-free culture. Nat Protoc 2007;2 (5):1197-205.
[27] Gordon GJ, Coleman WB, Grisham JW. Temporal analysis of hepatocyte differentiation by small hepatocyte-like progenitor cells during liver regeneration in retrorsine-exposed rats. Am J Pathol 2000;157 (3):771-86.
[28] Gordon GJ, Coleman WB, Hixson DC, Grisham JW. Liver regeneration in rats with retrorsine-induced hepatocellular injury proceeds through a novel cellular response. Am J Pathol 2000;156 (2):607-19.
[29] Malhi H, Irani AN, Gagandeep S, Gupta S. Isolation of human progenitor liver epithelial cells with extensive replication capacity and differentiation into mature hepatocytes. J Cell Sci 2002;115 (Pt 13):2679-88.
[30]苏娟,姚玉成,王忠华,王新民,马大烈,余宏宇,熊俊,訾晓渊,赵书民,胡以平.干细胞样原始肝细胞的分离和鉴定.癌变畸变突变2000;12(4):200-201.
[31] Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, Boggs SS, Greenberger JS, Goff JP. Bone marrow as a potential source of hepatic oval cells. Science 1999;284 (5417):1168-70.
[32]Avital I, Inderbitzin D, Aoki T, Tyan DB, Cohen AH, Ferraresso C, Rozga J, Arnaout WS, Demetriou AA. Isolation, characterization, and transplantation of bone marrow-derived hepatocyte stem cells. Biochem Biophys Res Commun 2001;288 (1):156-64.
[33] Avital I, Feraresso C, Aoki T, Hui T, Rozga J, Demetriou A, Muraca M. Bone marrow-derived liver stem cell and mature hepatocyte engraftment in livers undergoing rejection. Surgery 2002;132 (2):384-90.
[34] Inderbitzin D, Avital I, Gloor B, Keogh A, Candinas D. Functional comparison of bone marrow-derived liver stem cells: selection strategy for cell-based therapy. J Gastrointest Surg 2005;9 (9):1340-5.
[35] Harris JR, Brown GA, Jorgensen M, Kaushal S, Ellis EA, Grant MB, Scott EW. Bone marrow-derived cells home to and regenerate retinal pigment epithelium after injury. Invest Ophthalmol Vis Sci 2006;47 (5):2108-13.
[36] Inderbitzin D, Avital I, Keogh A, Beldi G, Quarta M, Gloor B, Candinas D. Interleukin-3 induces hepatocyte-specific metabolic activity in bone marrow-derived liver stem cells. J Gastrointest Surg 2005;9 (1):69-74.
[37] Dabeva MD, Hwang SG, Vasa SR, Hurston E, Novikoff PM, Hixson DC, Gupta S, Shafritz DA. Differentiation of pancreatic epithelial progenitor cells into hepatocytes following transplantation into rat liver. Proc Natl Acad Sci U S A 1997;94 (14):7356-61.
[38] Shen CN, Slack JM, Tosh D. Molecular basis of transdifferentiation of pancreas to liver. Nat Cell Biol 2000;2 (12):879-87.
[39] Hay DC, Zhao D, Ross A, Mandalam R, Lebkowski J, Cui W. Direct differentiation of human embryonic stem cells to hepatocyte-like cells exhibiting functional activities. Cloning Stem Cells 2007;9 (1):51-62.
[40] Weber A. Immortalization of hepatic progenitor cells. Pathol Biol (Paris) 2004;52 (2):93-6.
[41] Kamiya A, Gonzalez FJ, Nakauchi H. Identification and differentiation of hepatic stem cells during liver development. Front Biosci 2006;11:1302-10.
[42] Grisham JW, Coleman WB, Smith GJ. Isolation, culture, and transplantation of rat hepatocytic precursor (stem-like) cells. Proc Soc Exp Biol Med 1993;204 (3):270-9.
[43] Nagai H, Terada K, Watanabe G, Ueno Y, Aiba N, Shibuya T, Kawagoe M, Kameda T, Sato M, Senoo H, Sugiyama T. Differentiation of liver epithelial (stem-like) cells into hepatocytes induced by coculture with hepatic stellate cells. Biochem Biophys Res Commun 2002;293 (5):1420-5.
[44] Germain L, Noel M, Gourdeau H, Marceau N. Promotion of growth and differentiation of rat ductular oval cells in primary culture. Cancer Res 1988;48 (2):368-78.
[45] Lazaro CA, Rhim JA, Yamada Y, Fausto N. Generation of hepatocytes from oval cell precursors in culture. Cancer Res 1998;58 (23):5514-22.
[46] Jung Y, Oh SH, Zheng D, Shupe TD, Witek RP, Petersen BE. A potential role of somatostatin and its receptor SSTR4 in the migration of hepatic oval cells. Lab Invest 2006;86 (5):477-89.
[47] Yang L, Li S, Hatch H, Ahrens K, Cornelius JG, Petersen BE, Peck AB. In vitro trans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells. Proc Natl Acad Sci U S A 2002;99 (12):8078-83.
[48] Oertel M, Menthena A, Dabeva MD, Shafritz DA. Cell competition leads to a high level of normal liver reconstitution by transplanted fetal liver stem/progenitor cells. Gastroenterology 2006;130 (2):507-20; quiz 90.
[49] Lemmer ER, Vessey CJ, Gelderblom WC, Shephard EG, Van Schalkwyk DJ, Van Wijk RA, Marasas WF, Kirsch RE, Hall Pde L. Fumonisin B1-induced hepatocellular and cholangiocellular tumors in male Fischer 344 rats: potentiating effects of 2-acetylaminofluorene on oval cell proliferation and neoplastic development in a discontinued feeding study. Carcinogenesis 2004;25 (7):1257-64.
[50] Sharma AD, Cantz T, Manns MP, Ott M. The role of stem cells in physiology, pathophysiology, and therapy of the liver. Stem Cell Rev 2006;2 (1):51-8.
[51] Baumann U, Crosby HA, Ramani P, Kelly DA, Strain AJ. Expression of the stem cell factor receptor c-kit in normal and diseased pediatric liver: identification of a human hepatic progenitor cell? Hepatology 1999;30 (1):112-7.
[52] Dumble ML, Croager EJ, Yeoh GC, Quail EA. Generation and characterization of p53 null transformed hepatic progenitor cells: oval cells give rise to hepatocellular carcinoma. Carcinogenesis 2002;23 (3):435-45.
[53] Dumble ML, Knight B, Quail EA, Yeoh GC. Hepatoblast-like cells populate the adult p53 knockout mouse liver: evidence for a hyperproliferative maturation-arrested stem cell compartment. Cell Growth Differ 2001;12 (5):223-31.
[54] Lowes KN, Brennan BA, Yeoh GC, Olynyk JK. Oval cell numbers in human chronic liver diseases are directly related to disease severity. Am J Pathol 1999;154 (2):537-41.
[55] Sell S, Salman J. Light- and electron-microscopic autoradiographic analysis of proliferating cells during the early stages of chemical hepatocarcinogenesis in the rat induced by feeding N-2-fluorenylacetamide in a choline-deficient diet. Am J Pathol 1984;114 (2):287-300.
[56] Li YG, Chen M, Zhang DZ, Wang ZY, Zeng WQ, Shi XF, Guo Y, Guo SH, Ren H. [Clinical research on the treatment effect of autologous dendritic cell vaccine on the patients with chronic hepatitis B]. Zhonghua Gan Zang Bing Za Zhi 2003;11 (4):206-8.
[57] Chen M, Li YG, Zhang DZ, Wang ZY, Zeng WQ, Shi XF, Guo Y, Guo SH, Ren H. Therapeutic effect of autologous dendritic cell vaccine on patients with chronic hepatitis B: a clinical study. World J Gastroenterol 2005;11 (12):1806-8.
[58] Cai DC, Li J, Zeng Y, Li YG, Ren H. [A study on the anti-HBV effect of dendritic cell from human umbilical cord blood]. Zhonghua Gan Zang Bing Za Zhi 2007;15 (2):88-91.
[59] Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin 2005;55 (2):74-108.
[60] Bergsland EK. Molecular mechanisms underlying the development of hepatocellular carcinoma. Semin Oncol 2001;28 (5):521-31.
[61] Thorgeirsson SS, Grisham JW. Molecular pathogenesis of human hepatocellular carcinoma. Nat Genet 2002;31 (4):339-46.
[62] Satyanarayana A, Manns MP, Rudolph KL. Telomeres and telomerase: a dual role in hepatocarcinogenesis. Hepatology 2004;40 (2):276-83.
[63] Brechot C. Pathogenesis of hepatitis B virus-related hepatocellular carcinoma: old and new paradigms. Gastroenterology 2004;127 (5 Suppl 1):S56-61.
[64] Velazquez RF, Rodriguez M, Navascues CA, Linares A, Perez R, Sotorrios NG, Martinez I, Rodrigo L. Prospective analysis of risk factors for hepatocellular carcinoma in patients with liver cirrhosis. Hepatology 2003;37 (3):520-7.
[65] Hassan MM, Hwang LY, Hatten CJ, Swaim M, Li D, Abbruzzese JL, Beasley P, Patt YZ. Risk factors for hepatocellular carcinoma: synergism of alcohol with viral hepatitis and diabetes mellitus. Hepatology 2002;36 (5):1206-13.
[66] Ming L, Thorgeirsson SS, Gail MH, Lu P, Harris CC, Wang N, Shao Y, Wu Z, Liu G, Wang X, Sun Z. Dominant role of hepatitis B virus and cofactor role of aflatoxin in hepatocarcinogenesis in Qidong, China. Hepatology 2002;36 (5):1214-20.
[67] Ohata K, Hamasaki K, Toriyama K, Matsumoto K, Saeki A, Yanagi K, Abiru S, Nakagawa Y, Shigeno M, Miyazoe S, Ichikawa T, Ishikawa H, Nakao K, Eguchi K. Hepatic steatosis is a risk factor for hepatocellular carcinoma in patients with chronic hepatitis C virus infection. Cancer 2003;97 (12):3036-43.
[68] Yano Y, Yamashita F, Sumie S, Ando E, Fukumori K, Kiyama M, Oyama T, Kuroki S, Kato O, Yamamoto H, Tanaka M, Sata M. Clinical features of hepatocellular carcinoma seronegative for both HBsAg and anti-HCV antibody but positive for anti-HBc antibody in Japan. Am J Gastroenterol 2002;97 (1):156-61.
[69] Pollicino T, Squadrito G, Cerenzia G, Cacciola I, Raffa G, Craxi A, Farinati F, Missale G, Smedile A, Tiribelli C, Villa E, Raimondo G. Hepatitis B virus maintains its pro-oncogenic properties in the case of occult HBV infection. Gastroenterology 2004;126 (1):102-10.
[70] Lok AS, McMahon BJ. Chronic hepatitis B: update of recommendations. Hepatology 2004;39 (3):857-61.
[71] Van Damme P, Van Herck K. A review of the efficacy, immunogenicity and tolerability of a combined hepatitis A and B vaccine. Expert Rev Vaccines 2004;3 (3):249-67.
[72]梁晓峰,陈园生,王晓军,贺雄,陈丽娟,王骏,林长缨,白呼群,严俊,崔钢,于竞进.中国3岁以上人群乙型肝炎血清流行病学研究.中华流行病学杂志2005,26(9):655-658.
[73]中华医学会第十二次全国病毒性肝炎及肝病学术会议纪要.中华传染病杂志2005,25(4): 286-287.
[74] Paterlini-Brechot P, Saigo K, Murakami Y, Chami M, Gozuacik D, Mugnier C, Lagorce D, Brechot C. Hepatitis B virus-related insertional mutagenesis occurs frequently in human liver cancers and recurrently targets human telomerase gene. Oncogene 2003;22 (25):3911-6.
[75] Murakami Y, Saigo K, Takashima H, Minami M, Okanoue T, Brechot C, Paterlini-Brechot P. Large scaled analysis of hepatitis B virus (HBV) DNA integration in HBV related hepatocellular carcinomas. Gut 2005;54(8):1162-8.
[76] Toyoda H, Kumada T, Kaneoka Y, Murakami Y. Impact of hepatitis B virus (HBV) X gene integration in liver tissue on hepatocellular carcinoma development in serologically HBV-negative chronic hepatitis C patients. J Hepatol 2008;48 (1):43-50.
[77] Herath NI, Leggett BA, MacDonald GA. Review of genetic and epigenetic alterations in hepatocarcinogenesis. J Gastroenterol Hepatol 2006;21 (1 Pt 1):15-21.
[78] Cougot D, Neuveut C, Buendia MA. HBV induced carcinogenesis. J Clin Virol 2005;34 Suppl 1:S75-8.
[79] Cui F, Wang Y, Wang J, Wei K, Hu J, Liu F, Wang H, Zhao X, Zhang X, Yang X. The up-regulation of proteasome subunits and lysosomal proteases in hepatocellular carcinomas of the HBx gene knockin transgenic mice. Proteomics 2006;6 (2):498-504.
[80] Wu CG, Forgues M, Siddique S, Farnsworth J, Valerie K, Wang XW. SAGE transcript profiles of normal primary human hepatocytes expressing oncogenic hepatitis B virus X protein. Faseb J 2002;16 (12):1665-7.
[81] Block TM, Mehta AS, Fimmel CJ, Jordan R. Molecular viral oncology of hepatocellular carcinoma. Oncogene 2003;22 (33):5093-107.
[82] Dewantoro O, Gani RA, Akbar N. Hepatocarcinogenesis in viral Hepatitis B infection: the role of HBx and p53. Acta Med Indones 2006;38 (3):154-9.
[83] Ahn JY, Chung EY, Kwun HJ, Jang KL. Transcriptional repression of p21(waf1) promoter by hepatitis B virus X protein via a p53-independent pathway. Gene 2001;275 (1):163-8.
[84] Tang H, Da L, Mao Y, Li Y, Li D, Xu Z, Li F, Wang Y, Tiollais P, Li T, Zhao M. Hepatitis B virus X protein sensitizes cells to starvation-inducedautophagy via up-regulation of beclin 1 expression. Hepatology 2009;49 (1):60-71.
[85] Zhang H, Shan CL, Li N, Zhang X, Zhang XZ, Xu FQ, Zhang S, Qiu LY, Ye LH, Zhang XD. Identification of a natural mutant of HBV X protein truncated 27 amino acids at the COOH terminal and its effect on liver cell proliferation. Acta Pharmacol Sin 2008;29 (4):473-80.
[86] Zhang X, Dong N, Yin L, Cai N, Ma H, You J, Zhang H, Wang H, He R, Ye L. Hepatitis B virus X protein upregulates survivin expression in hepatoma tissues. J Med Virol 2005;77 (3):374-81.
[87] Zhang X, Dong N, Zhang H, You J, Wang H, Ye L. Effects of hepatitis B virus X protein on human telomerase reverse transcriptase expression and activity in hepatoma cells. J Lab Clin Med 2005;145 (2):98-104.
[88] Lee YI, Han YJ, Lee SY, Lee YI, Park SK, Park YJ, Moon HB, Shin JH, Lee JH. Activation of insulin-like growth factor II signaling by mutant type p53: physiological implications for potentiation of IGF-II signaling by p53 mutant 249. Mol Cell Endocrinol 2003;203 (1-2):51-63.
[89] Fan ZR, Yang DH, Cui J, Qin HR, Huang CC. Expression of insulin like growth factor II and its receptor in hepatocellular carcinogenesis. World J Gastroenterol 2001;7 (2):285-8.
[90] Alexia C, Fourmatgeat P, Delautier D, Groyer A. Insulin-like growth factor-I stimulates H4II rat hepatoma cell proliferation: dominant role of PI-3'K/Akt signaling. Exp Cell Res 2006;312 (7):1142-52.
[91] Wei W, Huang W, Pan Y, Zhu F, Wu J. Functional switch of viral protein HBx on cell apoptosis, transformation, and tumorigenesis in association with oncoprotein Ras. Cancer Lett 2006;244 (1):119-28.
[92] Huang J, Wu K, Zhang J, Si W, Zhu Y, Wu J. Putative tumor suppressorYueF affects the functions of hepatitis B virus X protein in hepatoma cell apoptosis and p53 expression. Biotechnol Lett 2008;30 (2):235-42.
[93] Shih WL, Kuo ML, Chuang SE, Cheng AL, Doong SL. Hepatitis B virus X protein inhibits transforming growth factor-beta -induced apoptosis through the activation of phosphatidylinositol 3-kinase pathway. J Biol Chem 2000;275 (33):25858-64.
[94] Yoo YG, Lee MO. Hepatitis B virus X protein induces expression of Fas ligand gene through enhancing transcriptional activity of early growth response factor. J Biol Chem 2004;279 (35):36242-9.
[95] Yi YS, Park SG, Byeon SM, Kwon YG, Jung G. Hepatitis B virus X protein induces TNF-alpha expression via down-regulation of selenoprotein P in human hepatoma cell line, HepG2. Biochim Biophys Acta 2003;1638 (3):249-56.
[96] Kim KH, Seong BL. Pro-apoptotic function of HBV X protein is mediated by interaction with c-FLIP and enhancement of death-inducing signal. Embo J 2003;22 (9):2104-16.
[97] Chami M, Ferrari D, Nicotera P, Paterlini-Brechot P, Rizzuto R. Caspase-dependent alterations of Ca2+ signaling in the induction of apoptosis by hepatitis B virus X protein. J Biol Chem 2003;278 (34):31745-55.
[98] Chisari FV, Pinkert CA, Milich DR, Filippi P, McLachlan A, Palmiter RD, Brinster RL. A transgenic mouse model of the chronic hepatitis B surface antigen carrier state. Science 1985;230 (4730):1157-60.
[99] Xiong J, Yao YC, Zi XY, Li JX, Wang XM, Ye XT, Zhao SM, Yan YB, Yu HY, Hu YP. Expression of hepatitis B virus X protein in transgenic mice. World J Gastroenterol 2003;9 (1):112-6.
[100]Gripon P, Diot C, Theze N, Fourel I, Loreal O, Brechot C, Guguen-Guillouzo C. Hepatitis B virus infection of adult human hepatocytes cultured in the presence of dimethyl sulfoxide. J Virol 1988;62 (11):4136-43.
[101]Diot C, Gripon P, Rissel M, Guguen-Guillouzo C. Replication of hepatitis B virus in differentiated adult rat hepatocytes transfected with cloned viral DNA. J Med Virol 1992;36 (2):93-100.
[102]Wu CH, Ouyang EC, Walton C, Promrat K, Forouhar F, Wu GY. Hepatitis B virus infection of transplanted human hepatocytes causes a biochemical and histological hepatitis in immunocopetentent rats. World J Gastroenterol 2003;9 (5):978-83.
[103]蒋黎,毛青,王宇明,李俊刚,刘俊. HBV感染的人鼠嵌合肝动物模型的建立.第三军医大学学报2003,25(5):120-122.
[104]谭龙益,叶廷军,王皓,孔宪涛;乙型肝炎病毒基因注入大鼠肝脏后的短暂抗原表达.第二军医大学学报1998,16(2):73-75.
[105]姚云清,张定凤,罗云,张大志,黄爱龙,王波,周卫平,任红,郭树华.乙型肝炎病毒裸DNA转染原代大鼠肝细胞模型的建立.中华肝脏病杂志2002,10(4); 36-40.
[106]于恒超李韧千年松窦科峰.大鼠肝硬化模型中小肝细胞分离培养方法的建立及其表型分析细胞与分子免疫学杂志2007;23(12):1203-1204.
[107]Zheng Y, Chen WL, Louie SG, Yen TS, Ou JH. Hepatitis B virus promotes hepatocarcinogenesis in transgenic mice. Hepatology 2007;45 (1):16-21.
[108]Zhu H, Wang Y, Chen J, Cheng G, Xue J. Transgenic mice expressing hepatitis B virus X protein are more susceptible to carcinogen induced hepatocarcinogenesis. Exp Mol Pathol 2004;76 (1):44-50.
[109]Slagle BL, Lee TH, Medina D, Finegold MJ, Butel JS. Increasedsensitivity to the hepatocarcinogen diethylnitrosamine in transgenic mice carrying the hepatitis B virus X gene. Mol Carcinog 1996;15 (4):261-9.
[110]Reid LM, Jefferson DM. Culturing hepatocytes and other differentiated cells. Hepatology 1984;4 (3):548-59.
[111]Runge D, Runge DM, Jager D, Lubecki KA, Beer Stolz D, Karathanasis S, Kietzmann T, Strom SC, Jungermann K, Fleig WE, Michalopoulos GK. Serum-free, long-term cultures of human hepatocytes: maintenance of cell morphology, transcription factors, and liver-specific functions. Biochem Biophys Res Commun 2000;269 (1):46-53.
[112]Fromm ME, Taylor LP, Walbot V. Stable transformation of maize after gene transfer by electroporation. Nature 1986;319 (6056):791-3.
[113] Nakamura T, Nawa K, Ichihara A. Partial purification and characterization of hepatocyte growth factor from serum of hepatectomized rats. Biochem Biophys Res Commun 1984;122 (3):1450-9.
[114]Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Largaespada DA, Verfaillie CM. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 2002;418 (6893):41-9.
[115]Shirakata Y, Kawada M, Fujiki Y, Sano H, Oda M, Yaginuma K, Kobayashi M, Koike K. The X gene of hepatitis B virus induced growth stimulation and tumorigenic transformation of mouse NIH3T3 cells. Jpn J Cancer Res 1989;80 (7):617-21.
[116]Luber B, Arnold N, Sturzl M, Hohne M, Schirmacher P, Lauer U, Wienberg J, Hofschneider PH, Kekule AS. Hepatoma-derived integrated HBV DNA causes multi-stage transformation in vitro. Oncogene 1996;12(8):1597-608.
[117]李涛,刘国珍,谭德明,吴传湘,刘洪波.乙肝病毒x基因真核表达质粒及细胞模型的建立.生命科学研究2007;11(4):355-360.
[118]Sell S, Xu KL, Huff WE, Kabena LF, Harvery RB, Dunsford HA. Aflatoxin exposure produces serum alphafetoprotein elevations and marked oval cell proliferation in young male Pekin ducklings. Pathology 1998;30 (1):34-9.
[119]Coleman WB, Wennerberg AE, Smith GJ, Grisham JW. Regulation of the differentiation of diploid and some aneuploid rat liver epithelial (stemlike) cells by the hepatic microenvironment. Am J Pathol 1993;142 (5):1373-82.
[120]Steinberg P, Steinbrecher R, Radaeva S, Schirmacher P, Dienes HP, Oesch F, Bannasch P. Oval cell lines OC/CDE 6 and OC/CDE 22 give rise to cholangio-cellular and undifferentiated carcinomas after transformation. Lab Invest 1994;71 (5):700-9.