HBV适应性杂交细胞株的建立和初步研究
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摘要
研究背景
乙型肝炎病毒(hepatitis B virus,HBV)是引起严重肝脏疾病的病原体之一。据世界卫生组织报道,全球20多亿人曾感染乙肝病毒,其中约3.5亿人为慢性乙肝病毒感染者。慢性乙肝不易彻底治愈,并随着病情发展可恶化成肝硬化、肝癌。自1965年Blumberg等发现乙型肝炎病毒表面抗原,到1997年病毒全基因组的克隆和测序完成,人们对HBV的基因组成和抗原的结构、生物特性及其生命周期的认识迅速发展。但因缺乏体外增殖系统和易获得的细胞模型,HBV感染肝脏的早期机理研究进展较为缓慢,从而在一定程度上影响乙型肝炎及其相关肝脏疾病的基础和临床研究。由于人的肝组织来源有限,而且HBV病毒粘附以及DNA转录和复制对宿主有严格的限制,先前研究过的细胞模型又各存在其不可避免的缺陷。因此,建立一个近于自然感染状态即既适用于转染又可用血清中病毒颗粒直接感染,接近肝细胞生理状态并能稳定表达HBV抗原的细胞模型是急需的。
本研究旨在通过将利用化学诱变剂诱导产生次黄嘌呤鸟嘌呤磷酸核糖转移酶(HGPRT)基因突变的HepG2细胞与人原代肝细胞融合,建立杂交细胞株,这个杂交细胞株从细胞基因工程学理论上讲应该具有双亲遗传学特性,即既具有人原代肝细胞对HBV易感的特性又具有HepG2细胞能体外长期传代的特性。并对HBV在该杂交细胞的感染情况进行初步研究。
目的
建立不同于HepG2细胞及人原代肝细胞的新型杂交细胞株,使该细胞株既能长期携带HBV又能体外传代培养。
方法
诱导HepG2细胞产生HGPRT基因突变,将筛选出的HGPRT缺陷的HepG2细胞与携带HBV的人原代肝细胞进行融合得杂交细胞,用HAT培养基筛选出异核体杂交细胞,再利用有限稀释法进行克隆,通过核型分析方法鉴定所得细胞。对所得杂交细胞及培养上清液进行HBV DNA和HBsAg、HBeAg的检测。实验同时,用未杂交的HepG2细胞和人原代肝细胞作对照。
结果
经筛选后,有一杂交细胞株(HepCHLine3)克隆成功。HepCHLine3在形态学上与HGPRT缺陷HepG2细胞相似,能体外传代培养,染色体核型分析示HepCHLine3杂交细胞染色体众数为99条,证明为融合细胞株,含所有来自HepG2细胞和人原代肝细胞的基因数。传代培养的HepCHLine3及其培养上清液用巢式PCR可分别检测到HBV DNA,培养上清液内检测到HBsAg和HBeAg。对照组的HepG2细胞和人原代肝细胞相应结果为阴性。结果提示该细胞携带并分泌HBV DNA。
结论
该杂交细胞兼具HepG2细胞体外传代和人原代肝细胞对HBV易感的特性,是一新型杂交细胞株,为进一步建立新型HBV感染细胞模型奠定了基础。
Background
The hepatitis B virus(HBV), is one of the major pathogens which can cause severe liver disease. It was reported by WHO, globally, more than 2 billion people were infected by HBV. And there are about 350 million chronic carriers of HBV. The chronic hepatitis B can not be cured, What's more, hepatitis B can deteriorate to cirrhosis and hepatocellular carcinoma with the development of illness. From Blumberg having found HBV surface antigen in 1965 to having completed clone and sequence of HBV DNA genome in 1997, researchers have been making great progresses in constitution of HBV genome, structure and biological traits of antigens, and life circle of HBV. Due to deficiency of exoteric multiplying system and pragmatic cell models, little was known about the early mechanisms of initiation in infection. Consequently, basic and clinical research on hepatitis B and other types of hepatitis concerned were hampered some way. Owing to the finiteness of human hepatic tissue, the restriction of HBV adhesion, DNA transcription and replication host, at the same time prior cell models are all have their inevitable defects, we need to establish a better cell model. This model should be both suitable for transfection and sensitive to virus particle from blood serum. The cell should be similar to human hepatic cell in morphology and can express HBV antigens stably.
Objective
To establish a hybrid cell that is different from either HepG2 cell or human primary hepatic cell. This cell carries HBV, and can be serial subcultivation in vitro.
Methods
HGPRT defect HepG2 cell, which was induced by 6-MP, was fused with human primary hepatic cell infected by hepatitis B virus. After being screened with HAT, the hybrid cell was cloned through limiting dilution assay. Subsequently, the hybrid cell was identified by karyotype analysis. HBV DNA and HBsAg、HBeAg were examined at different generations. HepG2 and human primary hepatic cell infected by hepatitis B virus were taken as control groups.
Results
The hybrid cell is similar to HGPRT defect HepG2 cell morphologically and can be subcultured in vitro. The Karyotype analysis results show that the modal chromosome numbers are 99 in the hybrid cells, indicating that the hybrid cells contain all genomic factors from both HepG2 and human hepatic cell. HBV DNA can be detected in the cells and supernates by nest PCR. In the supernates HBsAg and HBeAg can be detected too. Conceivably the hybrid cell possesses the replication a generation in vitro of HepG2 as well as the sensitivity of human primary hepatic cell to HBV, which paves secretion of HBV DNA.
Conclusion
As a new hybrid cell line, it has the characteristics of both generation in vitro of HepG2 and sensitivity of human primary hepatic cell to HBV, which paves way for further study of HBV infection cell model.
乙型肝炎病毒(hepatitis B virus,HBV)是引起严重肝脏疾病的病原体之一。据世界卫生组织报道,全球20多亿人曾感染乙肝病毒,其中约3.5亿人为慢性乙肝病毒感染者。慢性乙肝不易彻底治愈,并随着病情发展可恶化成肝硬化、肝癌。自1965年Blumberg等发现乙型肝炎病毒表面抗原,到1997年病毒全基因组的克隆和测序完成,人们对HBV的基因组成和抗原的结构、生物特性及其生命周期的认识迅速发展。但因缺乏体外增殖系统和易获得的细胞模型,HBV感染肝脏的早期机理研究进展较为缓慢,从而在一定程度上影响乙型肝炎及其相关肝脏疾病的基础和临床研究。由于人的肝组织来源有限,而且HBV病毒粘附以及DNA转录和复制对宿主有严格的限制,先前研究过的细胞模型又各存在其不可避免的缺陷。因此,建立一个近于自然感染状态即既适用于转染又可用血清中病毒颗粒直接感染,接近肝细胞生理状态并能稳定表达HBV抗原的细胞模型是急需的。
本研究旨在通过将利用化学诱变剂诱导产生次黄嘌呤鸟嘌呤磷酸核糖转移酶(HGPRT)基因突变的HepG2细胞与人原代肝细胞融合,建立杂交细胞株,这个杂交细胞株从细胞基因工程学理论上讲应该具有双亲遗传学特性,即既具有人原代肝细胞对HBV易感的特性又具有HepG2细胞能体外长期传代的特性。并对HBV在该杂交细胞的感染情况进行初步研究。
目的
建立不同于HepG2细胞及人原代肝细胞的新型杂交细胞株,使该细胞株既能长期携带HBV又能体外传代培养。
方法
诱导HepG2细胞产生HGPRT基因突变,将筛选出的HGPRT缺陷的HepG2细胞与携带HBV的人原代肝细胞进行融合得杂交细胞,用HAT培养基筛选出异核体杂交细胞,再利用有限稀释法进行克隆,通过核型分析方法鉴定所得细胞。对所得杂交细胞及培养上清液进行HBV DNA和HBsAg、HBeAg的检测。实验同时,用未杂交的HepG2细胞和人原代肝细胞作对照。
结果
经筛选后,有一杂交细胞株(HepCHLine3)克隆成功。HepCHLine3在形态学上与HGPRT缺陷HepG2细胞相似,能体外传代培养,染色体核型分析示HepCHLine3杂交细胞染色体众数为99条,证明为融合细胞株,含所有来自HepG2细胞和人原代肝细胞的基因数。传代培养的HepCHLine3及其培养上清液用巢式PCR可分别检测到HBV DNA,培养上清液内检测到HBsAg和HBeAg。对照组的HepG2细胞和人原代肝细胞相应结果为阴性。结果提示该细胞携带并分泌HBV DNA。
结论
该杂交细胞兼具HepG2细胞体外传代和人原代肝细胞对HBV易感的特性,是一新型杂交细胞株,为进一步建立新型HBV感染细胞模型奠定了基础。
Background
The hepatitis B virus(HBV), is one of the major pathogens which can cause severe liver disease. It was reported by WHO, globally, more than 2 billion people were infected by HBV. And there are about 350 million chronic carriers of HBV. The chronic hepatitis B can not be cured, What's more, hepatitis B can deteriorate to cirrhosis and hepatocellular carcinoma with the development of illness. From Blumberg having found HBV surface antigen in 1965 to having completed clone and sequence of HBV DNA genome in 1997, researchers have been making great progresses in constitution of HBV genome, structure and biological traits of antigens, and life circle of HBV. Due to deficiency of exoteric multiplying system and pragmatic cell models, little was known about the early mechanisms of initiation in infection. Consequently, basic and clinical research on hepatitis B and other types of hepatitis concerned were hampered some way. Owing to the finiteness of human hepatic tissue, the restriction of HBV adhesion, DNA transcription and replication host, at the same time prior cell models are all have their inevitable defects, we need to establish a better cell model. This model should be both suitable for transfection and sensitive to virus particle from blood serum. The cell should be similar to human hepatic cell in morphology and can express HBV antigens stably.
Objective
To establish a hybrid cell that is different from either HepG2 cell or human primary hepatic cell. This cell carries HBV, and can be serial subcultivation in vitro.
Methods
HGPRT defect HepG2 cell, which was induced by 6-MP, was fused with human primary hepatic cell infected by hepatitis B virus. After being screened with HAT, the hybrid cell was cloned through limiting dilution assay. Subsequently, the hybrid cell was identified by karyotype analysis. HBV DNA and HBsAg、HBeAg were examined at different generations. HepG2 and human primary hepatic cell infected by hepatitis B virus were taken as control groups.
Results
The hybrid cell is similar to HGPRT defect HepG2 cell morphologically and can be subcultured in vitro. The Karyotype analysis results show that the modal chromosome numbers are 99 in the hybrid cells, indicating that the hybrid cells contain all genomic factors from both HepG2 and human hepatic cell. HBV DNA can be detected in the cells and supernates by nest PCR. In the supernates HBsAg and HBeAg can be detected too. Conceivably the hybrid cell possesses the replication a generation in vitro of HepG2 as well as the sensitivity of human primary hepatic cell to HBV, which paves secretion of HBV DNA.
Conclusion
As a new hybrid cell line, it has the characteristics of both generation in vitro of HepG2 and sensitivity of human primary hepatic cell to HBV, which paves way for further study of HBV infection cell model.
引文
[1]Gao C,Mao S,Kaufmann G.et al.A method for the generation of combinatorial antibody libraries using pⅨ Phage display[J].Proc Natl Acad Sci USA,2002;99(20):12612-12616.
[2]杨国敏.核苷类似物治疗慢性乙肝临床应用与耐药预防[J].中国医刊,2007:42(11):64.
[3]Puviani AC,Tichonicky L,Glaise D.et al.Development of novel radial-flow bioreactor for biohybrid liver support system.New Frontiers in the Treatment of liver Diseases[J].December 1997;6.
[4]王俊明,牟振云.乙型肝炎病毒基因分型及其临床意义[J].临床荟萃,2006;21(11):825-826.
[5]鄂征主编.组织培养和分子细胞生物学技术[M].北京出版社:1997年2月第二版.
[6]Lefert H,Fuller BJ,Moore CJ.et al.Hormonal control of rat liver regeneration[J].Gastroenterology,1997;76:1470-1482.
[7]Nir Paran,Benjamin Geiger,Yosef Shaul.HBV infection of cell culture:evidence for multivalent and cooperative attachment[J].The EMBO Journal,2001;20(16):4443-4453.
[8]Mine T,Ketterer B,Meyer DJ,et al.Difference in sensitivity to glucagons action in three different rat liver systems[J].Metab clin Exp,1998;39:321-326.
[9]赵克开,缪晓辉,徐文胜.HepG2.2.15细胞内乙型肝炎病毒cccDNA的定量检测[J].中华传染病杂志,2005:23(1):6-9.
[10]Boyer JL,Guengerich FP,Distilerath LM.et al.Preparation and specific applications of isolated hepatocyte couplets[J].Methods Enzymol,1998;192:501-516.
[11]Jauregui HO,Chipman JK,Simpson JG.et al.Primary cultures of rat hepatocytes in hollow fiber chambers[J].In vitro Cell Dev Diol,1994;30A:23-49.
[12]Lee WM.Hepatitis B Virus infection[J].N.Engl.J.Med,1997;337:1733-1745.
[13]Laras A,Koskinas J,Dimou E.et al.Intrahepatic levels and replicative activity of covalently closed circular hepatitis B virus DNA in chronically infected patients[J].Hepatology,2006;44(3):694-702.
[14]Dienstag JL,Mabit H,Capel F.et al.Lamivudine as initial treatment for chronic hepatitis B in the United States[J].N.Engl.J.Med,1999;34:1256-1263.
[15]马巧玉,王宇明,郝飞等.体外分离培养正常成人肝细胞的方法[J].第三军医大学学报,2001:23(2):239-240.
[16]Mito M,Kjellin L,Laurent TC.et al.Hepatocyte transplantation in man[J].Cell Transplant,1993;2:65-74.
[17]韩聚强.体外肝细胞培养技术新进展[J].河北医科大学学报,2002:23(3):184-186.
[18]Walpole HE,Guillouzo A,Yarmush ML.et al.Rabbit hepatocytes in primary culture:preparation,viability and use in studies of propanolol metabolism[J].Hepatology,1994;11:394-401.
[19]Wiederkehr JC,Fuller BJ,Santone KS.et al.Hepatocyte transplantation for the low-density lipoprotein receptor-deficient state[J].Transplantation,1998;50:466-476.
[20]张莉萍,康格非.原代肝细胞培养的研究现状[J].国外医学临床生物化学与检验学分册,2004:25(3):193-196.
[21]Watanabe FD,Storm SC,Nutt LH.et al.Clinical experience with a bioartificial liver in the treatment of severe liver failure[J].Ann Surg,1997:225:484-494.
[22]Katenz E,Vondran FW,Schwartlander R.et al.Cryopreservation of primary human hepatocytes:the benefit of trehalose as an additional cryoprotective agent[J].Liver Transpl,2007;13(1):38-45.
[23]Xie ZC,Marion PL,Robinson WS.et al.Transmission of hepatitis C virus infection to trees hrews[J].Virology,1998;244:513-520.
[24]Nishimura M,Ueda N,Naito S.Effects of dimethyl sulfoxide on the gene induction of cytochrome P450 isoforms,UGT-dependent glucuronosyl transferase isoforms,and ABCB1 in primary culture of human hepatocytes[J].Biol Pharm Bull,2003;26(7):1052-1056.
[25]Josef K,Summers J,Hanada S.et al.Efficient Infection of primary tupaia hepatocytes with purified human and woolly monkey hepatitis B virus[J].Journal of virology,2001;11:5084-5089.
[26]Mc Caffrey AP,Pandey K,Pandey K.Inhibition of hepatitis B virus in mice by RNA interference[J].Nature Biotechnology,2003;21(6):639-644.
[27]Suzuki A,Blum HE,Haase AT.et al.Functional absence of FADD in PLC/PRF/5 hepatoma cells[J].Proc Soc Exp Biol Med,1999;221:72-79.
[28]Yared G,Blum HE,Zhang ZS.et al Cytokine-mediated apoptosis and inhibition of virus production and anchorage independent growth of viral transfected hepatoblastoma cells[J].Cytokine,1998;10:586-595.
[29]Lara P,Galum E,Sells MA.et al.The hepatitis B virus X protein up-regulates tumor necrosis factor alPHa gene expression in hepatocytes[J].Hepatology,1998;28:1013-1021.
[30]司徒镇强,吴军正.细胞培养[M].西安:世界图书出版公司,1996,181-187.
[31]崔向东.刘金刚.器官的低温生物学基础极其低温保存[M]//刘金刚.刘作斌.低温医学.北京:人民卫生出版社。1993:6-87.
[32]HENGSTLER J G,UTESCH D,STEINBERG P.et al.Cryopreserved primary hepatocytes as aconstantly available in vitro model for the evaluation of human and animal drugmetabolism and enzyme induction[J].Drug Metabolism Rev,2000;32(1):81-118.
[33] NAIK S, SANTANGINI H A, TRENKLER D M. et al. Functional recovery of porcine hepatocytes after hypothermic or cryogenic preservation for liver support systems[J]. Cell Transplant, 1997;6(5): 447-454.
[34] GUILLOUZO A, RIALLAND L, FAUTREL A. et al. Survival and function of isolated hepatocytes after cryopreservation[J]. Chem Boil Interact, 1999:121(1): 7-16.
[35] Rumin S, Gripon P, Seyec JL, Corral-Debrinski M. et al. Long-term productive episomal hepatitis B virus replication in primary cultures of adult human hepatocytes infected in vitro[J]. Virol Hepatol, 1996:3:227-238.
[36] 刘权章.人类染色体方法学[M]. 北京:人民卫生出版社, 1992,273-274.
[37] Loeb DD, Tian R, Gualya AE. Changing the site of initation of plus-strand DNA synthesis inhibits the subsequent template switch during replication of hepadnavirus[J]. Virol, 1998;72:6565-6573.
[38] Fallows DA, Goff SP. Mutations in the epsilon sequences of human hepatitis B virus affect both RNA encapsidation and reverse transcription[J]. Virol, 1995;69:3067-3073.
[39] Tang H, McLachlan A. Transcription regulation of hepatitis B virus by nuclear hormone receptors is a critical determinant of viral tropism[J]. Proc Natl Acad Sci USA,2001; 98(4): 1841.
[40] Ryu WS. Aspects of hepatitis B viral infection and the viral carcinogenesis[J]. Biochem and M01 Biology, 2003:36:138-143.
[41] Rijntjes PJ, Moshage HJ. In vitro infection of primary cultures of cryopreserved adult human hepatocytes with hepatitis B virus[J]. Virus Res, 1998;10(1):95-109.
[42] Ochiya T, Tsurimoto T, Ueda K. An in vitro system for infection with hepatitis B virus that uses primary human fetal hepatocytes[J]. Proc Natl Acad Sci USA, 1989;86(6):1875-1879.
[43] Philippe G, Sylvie R, Stephan U, et al. Infection of a human hepatoma cell line by hepatitis B virus[J].Proc Natl Acad Sci USA,2002;99(24):15655-15660.
[44]Sprinzl MF,Oberwinkler H,Schaller H,et al.Transfer of hepatitis B virus genome by adenovirus vectors into cultured cells and mice:crossing the species barrier[J].Virol,2001;75(11):5108.
[45]Delaney WE,Isom HC.Hepatitis B virus replication in human HepG2cells mediated by hepatitis B virus recombinant baculovirus[J].Hepatology,1998;28:1134.
[46]Kitabwalla M.et al.N Engl J Med,2002;347(17):1364-1367.
[47]于德敏.HBV细胞模型及其在抗病毒药物研究中的应用[J].国外医学·流行病学传染病学分册,2003:30(4):213.
[48]高海燕.乙型肝炎病毒相关模型研究进展[J].泸州医学院学报,2007:30(1):66.
[49]Seifer M.Heermann KH,Gerlich WH.Replication of hepatitis B virus in transfected nonhepatic cell[J].Virology,1990:179(1):300.
[50]DIETER G,MEHRIAR A I,PERTER K.et al.Pre-S1 antigen-dependent infection of tupaia hepatocyte cultures with human hepatitis B virus[J].Virology,2003;77(17):9511-9521.
[51]Knowles BB,Howe CC,Aden DP.Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen[J].Science,1980;209(4455):497-499.
[52]王安辉,门可,张景霞等.HBV阳性血清体外感染HepG_2细胞的实验研究[J].第四军医大学学报,2003:24(24):2287-2289.
[2]杨国敏.核苷类似物治疗慢性乙肝临床应用与耐药预防[J].中国医刊,2007:42(11):64.
[3]Puviani AC,Tichonicky L,Glaise D.et al.Development of novel radial-flow bioreactor for biohybrid liver support system.New Frontiers in the Treatment of liver Diseases[J].December 1997;6.
[4]王俊明,牟振云.乙型肝炎病毒基因分型及其临床意义[J].临床荟萃,2006;21(11):825-826.
[5]鄂征主编.组织培养和分子细胞生物学技术[M].北京出版社:1997年2月第二版.
[6]Lefert H,Fuller BJ,Moore CJ.et al.Hormonal control of rat liver regeneration[J].Gastroenterology,1997;76:1470-1482.
[7]Nir Paran,Benjamin Geiger,Yosef Shaul.HBV infection of cell culture:evidence for multivalent and cooperative attachment[J].The EMBO Journal,2001;20(16):4443-4453.
[8]Mine T,Ketterer B,Meyer DJ,et al.Difference in sensitivity to glucagons action in three different rat liver systems[J].Metab clin Exp,1998;39:321-326.
[9]赵克开,缪晓辉,徐文胜.HepG2.2.15细胞内乙型肝炎病毒cccDNA的定量检测[J].中华传染病杂志,2005:23(1):6-9.
[10]Boyer JL,Guengerich FP,Distilerath LM.et al.Preparation and specific applications of isolated hepatocyte couplets[J].Methods Enzymol,1998;192:501-516.
[11]Jauregui HO,Chipman JK,Simpson JG.et al.Primary cultures of rat hepatocytes in hollow fiber chambers[J].In vitro Cell Dev Diol,1994;30A:23-49.
[12]Lee WM.Hepatitis B Virus infection[J].N.Engl.J.Med,1997;337:1733-1745.
[13]Laras A,Koskinas J,Dimou E.et al.Intrahepatic levels and replicative activity of covalently closed circular hepatitis B virus DNA in chronically infected patients[J].Hepatology,2006;44(3):694-702.
[14]Dienstag JL,Mabit H,Capel F.et al.Lamivudine as initial treatment for chronic hepatitis B in the United States[J].N.Engl.J.Med,1999;34:1256-1263.
[15]马巧玉,王宇明,郝飞等.体外分离培养正常成人肝细胞的方法[J].第三军医大学学报,2001:23(2):239-240.
[16]Mito M,Kjellin L,Laurent TC.et al.Hepatocyte transplantation in man[J].Cell Transplant,1993;2:65-74.
[17]韩聚强.体外肝细胞培养技术新进展[J].河北医科大学学报,2002:23(3):184-186.
[18]Walpole HE,Guillouzo A,Yarmush ML.et al.Rabbit hepatocytes in primary culture:preparation,viability and use in studies of propanolol metabolism[J].Hepatology,1994;11:394-401.
[19]Wiederkehr JC,Fuller BJ,Santone KS.et al.Hepatocyte transplantation for the low-density lipoprotein receptor-deficient state[J].Transplantation,1998;50:466-476.
[20]张莉萍,康格非.原代肝细胞培养的研究现状[J].国外医学临床生物化学与检验学分册,2004:25(3):193-196.
[21]Watanabe FD,Storm SC,Nutt LH.et al.Clinical experience with a bioartificial liver in the treatment of severe liver failure[J].Ann Surg,1997:225:484-494.
[22]Katenz E,Vondran FW,Schwartlander R.et al.Cryopreservation of primary human hepatocytes:the benefit of trehalose as an additional cryoprotective agent[J].Liver Transpl,2007;13(1):38-45.
[23]Xie ZC,Marion PL,Robinson WS.et al.Transmission of hepatitis C virus infection to trees hrews[J].Virology,1998;244:513-520.
[24]Nishimura M,Ueda N,Naito S.Effects of dimethyl sulfoxide on the gene induction of cytochrome P450 isoforms,UGT-dependent glucuronosyl transferase isoforms,and ABCB1 in primary culture of human hepatocytes[J].Biol Pharm Bull,2003;26(7):1052-1056.
[25]Josef K,Summers J,Hanada S.et al.Efficient Infection of primary tupaia hepatocytes with purified human and woolly monkey hepatitis B virus[J].Journal of virology,2001;11:5084-5089.
[26]Mc Caffrey AP,Pandey K,Pandey K.Inhibition of hepatitis B virus in mice by RNA interference[J].Nature Biotechnology,2003;21(6):639-644.
[27]Suzuki A,Blum HE,Haase AT.et al.Functional absence of FADD in PLC/PRF/5 hepatoma cells[J].Proc Soc Exp Biol Med,1999;221:72-79.
[28]Yared G,Blum HE,Zhang ZS.et al Cytokine-mediated apoptosis and inhibition of virus production and anchorage independent growth of viral transfected hepatoblastoma cells[J].Cytokine,1998;10:586-595.
[29]Lara P,Galum E,Sells MA.et al.The hepatitis B virus X protein up-regulates tumor necrosis factor alPHa gene expression in hepatocytes[J].Hepatology,1998;28:1013-1021.
[30]司徒镇强,吴军正.细胞培养[M].西安:世界图书出版公司,1996,181-187.
[31]崔向东.刘金刚.器官的低温生物学基础极其低温保存[M]//刘金刚.刘作斌.低温医学.北京:人民卫生出版社。1993:6-87.
[32]HENGSTLER J G,UTESCH D,STEINBERG P.et al.Cryopreserved primary hepatocytes as aconstantly available in vitro model for the evaluation of human and animal drugmetabolism and enzyme induction[J].Drug Metabolism Rev,2000;32(1):81-118.
[33] NAIK S, SANTANGINI H A, TRENKLER D M. et al. Functional recovery of porcine hepatocytes after hypothermic or cryogenic preservation for liver support systems[J]. Cell Transplant, 1997;6(5): 447-454.
[34] GUILLOUZO A, RIALLAND L, FAUTREL A. et al. Survival and function of isolated hepatocytes after cryopreservation[J]. Chem Boil Interact, 1999:121(1): 7-16.
[35] Rumin S, Gripon P, Seyec JL, Corral-Debrinski M. et al. Long-term productive episomal hepatitis B virus replication in primary cultures of adult human hepatocytes infected in vitro[J]. Virol Hepatol, 1996:3:227-238.
[36] 刘权章.人类染色体方法学[M]. 北京:人民卫生出版社, 1992,273-274.
[37] Loeb DD, Tian R, Gualya AE. Changing the site of initation of plus-strand DNA synthesis inhibits the subsequent template switch during replication of hepadnavirus[J]. Virol, 1998;72:6565-6573.
[38] Fallows DA, Goff SP. Mutations in the epsilon sequences of human hepatitis B virus affect both RNA encapsidation and reverse transcription[J]. Virol, 1995;69:3067-3073.
[39] Tang H, McLachlan A. Transcription regulation of hepatitis B virus by nuclear hormone receptors is a critical determinant of viral tropism[J]. Proc Natl Acad Sci USA,2001; 98(4): 1841.
[40] Ryu WS. Aspects of hepatitis B viral infection and the viral carcinogenesis[J]. Biochem and M01 Biology, 2003:36:138-143.
[41] Rijntjes PJ, Moshage HJ. In vitro infection of primary cultures of cryopreserved adult human hepatocytes with hepatitis B virus[J]. Virus Res, 1998;10(1):95-109.
[42] Ochiya T, Tsurimoto T, Ueda K. An in vitro system for infection with hepatitis B virus that uses primary human fetal hepatocytes[J]. Proc Natl Acad Sci USA, 1989;86(6):1875-1879.
[43] Philippe G, Sylvie R, Stephan U, et al. Infection of a human hepatoma cell line by hepatitis B virus[J].Proc Natl Acad Sci USA,2002;99(24):15655-15660.
[44]Sprinzl MF,Oberwinkler H,Schaller H,et al.Transfer of hepatitis B virus genome by adenovirus vectors into cultured cells and mice:crossing the species barrier[J].Virol,2001;75(11):5108.
[45]Delaney WE,Isom HC.Hepatitis B virus replication in human HepG2cells mediated by hepatitis B virus recombinant baculovirus[J].Hepatology,1998;28:1134.
[46]Kitabwalla M.et al.N Engl J Med,2002;347(17):1364-1367.
[47]于德敏.HBV细胞模型及其在抗病毒药物研究中的应用[J].国外医学·流行病学传染病学分册,2003:30(4):213.
[48]高海燕.乙型肝炎病毒相关模型研究进展[J].泸州医学院学报,2007:30(1):66.
[49]Seifer M.Heermann KH,Gerlich WH.Replication of hepatitis B virus in transfected nonhepatic cell[J].Virology,1990:179(1):300.
[50]DIETER G,MEHRIAR A I,PERTER K.et al.Pre-S1 antigen-dependent infection of tupaia hepatocyte cultures with human hepatitis B virus[J].Virology,2003;77(17):9511-9521.
[51]Knowles BB,Howe CC,Aden DP.Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen[J].Science,1980;209(4455):497-499.
[52]王安辉,门可,张景霞等.HBV阳性血清体外感染HepG_2细胞的实验研究[J].第四军医大学学报,2003:24(24):2287-2289.