适宜于HBV感染的新型细胞模型的研究
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摘要
研究背景
乙型肝炎病毒(hepatitis B virus,HBV)是嗜肝DNA病毒属的一员。HBV可以引起肝脏的急、慢性感染,HBV的慢性感染是引起严重肝脏疾病(比如:肝硬化、肝细胞肝癌)的主要病因。据世界卫生组织报道,全球20多亿人曾感染乙肝病毒,其中3.5亿以上为慢性乙型肝炎病毒感染者。慢性乙型肝炎不易彻底治愈,并且具有发展成肝硬化、肝癌的高度危险性。每年全世界范围内约有一百万人死于慢性乙型肝炎感染所致的肝衰竭、肝硬化和肝细胞肝癌。我国约有超过50%的居民曾被乙肝病毒感染过,其中8%～15%的人成为慢性感染者。
自从1965年Blumberg等发现乙型肝炎病毒表面抗原,到1997年乙肝病毒全基因组的克隆和测序完成,人们对HBV的基因组成、抗原结构、生物学特性及其生命周期的认识迅速发展。但是由于嗜肝DNA病毒属的感染具有严格的宿主特异性和组织特异性,限制了适合于研究人HBV感染的体外细胞模型的建立。
目前,用于体外研究HBV的细胞模型主要是人原代肝细胞和肝癌细胞来源的细胞系。人原代肝细胞用于HBV感染研究有其特有的优势:人原代肝细胞保留了分化肝细胞的重要细胞学特性,允许HBV的自然穿入和完整复制。然而,其缺陷在于在体外培养几天后,原代肝细胞开始出现肝细胞特殊形态的消失,并且失去了对HBV的感染和复制能力。此外,人原代肝细胞必须由新鲜肝脏组织获得,来源有限,并且其对病毒的摄取能力还受到细胞生长状态的影响。人原代肝细胞培养因为存在上述困难而限制了其应用,对HBV的研究更多的依赖于转染了HBV DNA克隆的分化良好的肝癌细胞系。其中最常用的是HepG2.2.15细胞系,HepG2.2.15细胞稳定转染了完整的HBV基因组,表达HBV RNA、病毒蛋白并能产生病毒样颗粒。由HepG2.2.15细胞分泌的病毒颗粒已经被证实可以感染黑猩猩。目前我们对于HBV复制和病毒基因组组成的许多重要的知识都是通过对转染了HBV DNA的肝癌细胞系的研究获得的。但是,由于HBV基因组是通过转染而非自然感染的方式进入到细胞中的,这些转染了HBV DNA的肝癌细胞系不能用于对HBV感染宿主细胞的早期感染过程的研究,包括病毒吸附,穿入和脱壳等过程。
由于缺乏有效的体外病毒感染和复制模型系统,HBV感染肝脏的早期机制研究进展较为缓慢,从而在一定程度上影响了乙型肝炎及其相关肝脏疾病的基础和临床研究。本研究旨在建立一个适合于HBV自然感染,并能有效进行病毒复制的细胞模型,为HBV感染宿丰细胞的早期机制,以及病毒侵入人肝细胞后完整的复制过程的研究提供一个有用的研究工具。
具体实验内容如下:
第一部分杂交细胞株HepCHLine-4的建立
【目的】
建立人原代肝细胞与HepG2的杂交细胞,以作为后续实验研究的基础。
【方法】
1建立人原代肝细胞与HepG2的杂交细胞。
1.1利用化学诱变剂诱导HepG2细胞产生次黄嘌呤鸟嘌呤磷酸核糖转移酶(HGPRT)基因突变。
1.2人原代肝细胞的分离与培养,台盼蓝染色排除法确定肝细胞存活率。
1.3利用细胞融合技术,将筛选出的HGPRT缺陷的HepG2细胞(HGPRTˉHepG2细胞)与人原代肝细胞进行融合,经HAT培养基筛选出异核体杂交细胞,进而用有限稀释法进行单克隆。
1.4利用胰蛋白酶G显带技术对所得细胞进行核型分析。
1.5绘制杂交细胞生长曲线。
2对杂交细胞是否携带HBV基因进行检测。
因为本研究中融合成功的杂交细胞是用慢性乙型肝炎携带者的肝细胞与HepG2细胞融合所得,所以在进行HBV感染实验之前我们对杂交细胞是否携带HBV基因进行检测。
2.1合成HBV型特异性引物,用巢式PCR检测杂交细胞内及培养上清中是否存在HBVDNA。
2.2提取杂交细胞基因组DNA,绿豆核酸酶消化后,巢式PCR检测杂交细胞内是否存在HBV的复制中间产物HBV cccDNA。
2.3杂交细胞是否表达HBV特异性蛋白抗原:间接免疫荧光检测杂交细胞内是否有HBcAg的表达;电化学发光法检测杂交细胞培养上清中的HBsAg和HBeAg。
【结果】
1成功建立人原代肝细胞与HepG2的杂交细胞。
1.1经EMS诱导突变得到的HGPRTˉHepG2细胞能耐受6-MP但不能在HAT选择培养基中生长,说明HGPRTˉHepG2细胞中次黄嘌呤核苷酸焦磷酸化酶(IPP)完全缺失。
1.2人原代肝细胞经台盼蓝染色排除法确定存活率≥90%。
1.3将HGPRTˉHepG2细胞和分离得到的人原代肝细胞融合后,经HAT选择培养基筛选和单克隆后,得到一株杂交细胞克隆,命名为HepCHLine-4。HepCHLine-4细胞在形态学上与HGPRTˉHepG2细胞相似,能传代培养,传代一次约需3～5天,经冻存复苏后可继续生长。
1.4染色体核型分析结果显示野生型HepG2细胞、HGPRTˉHepG2细胞、HepCHLine-4细胞染色体众数分别为51条、53条和99条,证实HepCHLine-4是一杂交细胞株,为HGPRTˉHepG2细胞(53条染色体)和人原代肝细胞(46条染色体)的杂合体,包含所有来自HGPRTˉHepG2细胞和人原代肝细胞的基因,兼有这两种细胞的遗传特性。
1.5绘制杂交细胞生长曲线,结果显示杂交细胞HepCHLine-4符合传代细胞生长过程特点。
2杂交细胞自身不携带HBV基因。
2.1杂交细胞内及培养上清中均未检测到HBV DNA。
2.2杂交细胞内不存在HBV的复制中间产物HBV cccDNA。
2.3杂交细胞不能表达HBV特异性蛋白抗原:杂交细胞内没有检测到HBcAg的表达;杂交细胞培养上清中没有检测到HBsAg和HBeAg。
【结论】
1.成功建立了一个兼具有HepG2和人原代肝细胞遗传特性的杂交细胞株HepCHLine-4,可以进一步用来研究建立适宜于HBV感染的细胞模型。
2.杂交细胞HepCHLine-4虽然是由慢性乙型肝炎携带者肝细胞与HepG2细胞融合所得,但是并不携带HBV基因及HBV的复制中间产物,故可以用于HBV感染的实验研究。
第二部分评价杂交细胞株HepCHLine-4对HBV的自然感染能力
【目的】
用慢性乙型肝炎患者血清中的病毒颗粒自然感染杂交细胞,评价杂交细胞对HBV的自然感染能力,探讨杂交细胞株HepCHLine-4用作HBV感染细胞模型的可能性。
【方法】
1.用含HBV DNA的慢性乙型肝炎患者血清分别与杂交细胞HepCHLine-4和HepG2细胞共同孵育,感染细胞,HepG2细胞作为对照细胞。
2.用HBV型特异性引物,巢式PCR检测感染后细胞内HBV DNA合成及分泌情况。
3.提取感染后细胞基因组DNA,绿豆核酸酶消化后,巢式PCR检测感染后细胞内有无HBV复制中间产物HBV cccDNA。
4.感染后杂交细胞中HBV特异性蛋白合成及分泌能力的检测:间接免疫荧光检测感染后细胞内是否有HBcAg的表达;电化学发光法检测感染后细胞培养上清中的HBsAg和HBeAg。
5.电镜检查感染后细胞能否向培养上清中分泌HBV病毒颗粒。
【结果】
1.HBV感染后的第6天到第15天,HepCHLine-4细胞内和培养上清中均能检测到HBV DNA;而感染后的HepG2细胞内及培养上清中均未检测到HBV DNA。
2.HBV感染后的第5天到第10天,HepCHLine-4细胞内均可以检测到HBV的复制中间产物HBV cccDNA的存在;而感染后的HepG2细胞内未检测到HBVcccDNA。
3.(1)HBV感染后的第1天到第30天(30天之后没有再进行检测),HepCHLine-4细胞胞浆内HBcAg始终是阳性表达,间接免疫荧光染色呈胞浆弥漫性着色;细胞核内HBcAg的阳性颗粒从感染后第2天到第30天都可以观察到,阳性率约15%,间接免疫荧光染色后表现为细胞核部分着色;而感染后的HepG2细胞内未检测到HBcAg的表达。(2)HBV感染后的第6天到第15天,HepCHLine-4细胞持续分泌HBsAg到培养上清中,培养上清中检测出的HBsAg的滴度最低值为3.850IU/mL,最高值为89.590IU/mL(≥1为阳性):HBV感染后的第8天到第17天,HepCHLine-4细胞持续分泌HBeAg到培养上清中,培养上清中检测出的HBeAg的滴度最低值为1.000S/Co,最高值为1.690S/Co(≥1为阳性);而感染后的HepG2细胞培养上清中未检测到HBsAg和HBeAg。
4.电镜结果:收集HBV感染后第6天的HepCHLine-4细胞培养上清,超速离心后,沉淀用2%磷钨酸负染,JEM-1200EX透射电镜观察。电镜下可以见到少量直径42nm的双层壳状Dane颗粒和较多的直径22nm的小球型颗粒及丝状颗粒。而感染后第6天的HepG2细胞培养上清超速离心后的沉淀电镜检查结果为阴性。
【结论】
杂交细胞HepCHLine-4可以被慢性乙型肝炎患者血清中的HBV病毒自然感染,感染后病毒可以侵入杂交细胞并在杂交细胞内进行病毒基因的复制,形成复制中间产物HBV cccDNA;并且进一步进行病毒基因的表达,合成HBV特异性蛋白HBsAg、HBeAg和HBcAg;HBV感染后杂交细胞能合成子代病毒颗粒并分泌出细胞。
到目前为止,经过了一年多的冻存复苏和传代培养,杂交细胞HepCHLine-4已经传代50代次以上,细胞形态和生长特性没有明显改变,并且保持其对HBV的易感性。
总结
本研究结果证实,我们建立的杂交细胞株HepCHLine-4兼具有HepG2和人原代肝细胞的遗传特性。它一方面继承了人原代肝细胞对HBV的易感性,另一方面,继承了HepG2细胞能在体外传代培养的特性,克服了人原代肝细胞作为HBV感染细胞模型的缺陷。HepCHLine-4对HBV自然感染易感,并能在感染后启动有效的病毒复制,形成复制中间产物HBV cccDNA,合成HBV特异性抗原并能合成子代病毒颗粒分泌出细胞。因此,杂交细胞株HepCHLine-4可以作为适宜于HBV自然感染的细胞模型,进行HBV感染宿主细胞的早期机制,包括病毒黏附、穿入、脱壳等早期感染过程的研究和病毒侵入人肝细胞后完整的复制过程的研究。
Background
Hepatitis B virus(HBV) is a member of the Hepadnavirus family.HBV causes transient and chronic infections of the liver.Chronic infection with HBV is a leading cause of severe liver diseases,such as cirrhosis and hepatocellular carcinoma.More than 2 billion people alive today have been infected by HBV,and more than 350 million people are chronically infected.Patients chronically infected with HBV run the risk of developing cirrhosis and hepatocellular carcinoma in later life.Worldwide deaths from chronic liver diseases and hepatocellular carcinoma caused by HBV infection approximately 1 million each year.In China,more than 50%of the population has been infected by HBV,and 8%to 15%of them become chronically infected.
Since HBV surface antigen was found by Blumberg in 1965 to the complete cloning and sequencing of HBV DNA genome in 1997,our knowledge on genetic constitution,antigenic structure,biology feature and life cycle of HBV have been riched.But Hepadnavirus infection is of strictly host specific and tissue specific, which hampers the establishment of suitable cell model for studying HBV infection.
Currently,there are two main cell types used for the study of HBV in vitro, primary human hepatocytes and hepatoma-derived cell lines.Primary human hepatocytes for infection studies have the advantage that they retain important cellular characteristics of differentiated hepatocytes allowing natural penetration,and the full replication of HBV.However,difficulties arise with this system because within several days in culture the cells begin to dedifferentiate and lose the capability to support HBV infection and replication.In addition,these cells must be obtained from a fresh liver,and the response of viral uptake varies greatly according to condition of harvested cells.Because of the difficulties with primary cell cultures,HBV research has relied upon the development of well-differentiated human hepatoma cell lines transfected with cloned HBV DNA.One of these cell lines,HepG2.2.15,is stably transfected with the complete HBV genome,expressing HBV RNA,viral proteins, and secreting virus-like particles.The virus secreted by HepG2.2.15 has demonstrated infectivity in chimpanzees.Many important details of HBV replication and genomic organization have been discovered through the use of transfected hepatoma cell lines. However,because the genome is introduced into cells by transfection rather than infection,these cell lines cannot be used to study HBV-host cell infection processes such as viral attachment,penetration,and uncoating.
Due to deficiency of an in vitro system for productive viral infection and replication,little has been known about the mechanisms of the early stages in HBV infection.Thereby,basic and clinical research on hepatitis B and chronic liver diseases correlated with HBV infection were hampered to some extent.Here,we have attempted to establish an in vitro infection cell culture system,which is susceptible to HBV infection and suitable for replication ensued.It could be a useful tool for investigating the mechanisms of the early stages in HBV infection,and analyzing the in vivo process of viral infection and viral life cycle in human hepatocytes.
SectionⅠEstablishing a Hybrid Cell Line HepCHLine-4
【Objective】
To establish a hybrid cell line susceptible for HBV infection by fused HepG2 cells with primary human hepatocytes.
【Methods】
1 To establish a hybrid cell line by fused HepG2 cells with primary human hepatocytes.
1.1 HepG2 cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT),were induced by EMS.
1.2 Isolation and culture of human primary hepatocytes.Hepatocytes viability was defined as the ability of cells to exclude trypan blue dyeing.
1.3 HGPRT~- HepG2 cells were fused with human primary hepatocytes.After being selected with HAT medium and limiting dilution assayed,the hybrid cell was monocloned.
1.4 The modal chromosome numbers in hybrid cell was counted by the trypsin G-banding method.
2 Since the primary hepatocytes we used for cell fusion was come from a patient chronically infected with HBV,it is necessary to analyze whether HepCHLine-4 contains HBV genome prior to HBV infection experiment.
2.1 We analyzed intracellular HBV DNA and HBV DNA in cultivate supernants. HBV genome was amplified by nested PCR using the universal primers for the outer primers,followed by a mixture containing B and C type-specific inner primers.
2.2 We analyzed intracellular HBV cccDNA by nested PCR after HepCHLine-4 cell genome digested with mung bean nuclease.
2.3 We detected whether HepCHLine-4 cells expressed HBV specific antigens. HBcAg in HepCHLine-4 cell was analyzed by indirect immunofluorescence staining.HBsAg and HBeAg in the supernatants were identified by electrochemiluminescence.
【Results】
1 We establish a hybrid cell line successfully by fused HepG2 cells with primary human hepatocytes.
1.1 HepG2 cells deficient in HGPRT(HGPRT~- HepG2) were induced by EMS. HGPRT~- HepG2 cells were resistant to 6-MP but could not grow in HAT medium, indicating a complete defect of inosinic acid pyrophosphorylase(IPP).
1.2 Human primary hepatocytes viability was defined≧ 90%.
1.3 After cell fusion and being selected with HAT medium and limiting dilution assayed,we established a bybrid cell line,named HepCHLine-4.HepCHLine-4 was similar to HGPRT~- HepG2 cells morphologically,and can be subcultured in vitro.HepCHLine-4 cell line can be resuscitated after being freezed.
1.4 The Karyotype analysis results showed that the modal chromosome numbers of wild-type HepG2,HGPRT~- HepG2 and HepCHLine-4 were 51,53 and 99, respectively,indicating that the HepCHLine-4 was a hybrid cell line between HGPRT~- HepG2 cells(53 chromosomes) and human hepatocytes(46 chromosomes),and contained all genomic factors from both human hepatocytes and HepG2 cells.
2 The hybrid cell line doesn't contain HBV genome prior to HBV infection experiment.
2.1 HBV DNA could not be detected in HepCHLine-4 cells or in the cultivate supernants.
2.2 HBV cccDNA,the replicative intermediate of HBV,could not be detected in HepCHLine-4 cells.
2.3 HepCHLine-4 cells could not express HBV specific antigens prior to HBV infection.HBcAg could not be detected in HepCHLine-4 cells.HBsAg and HBeAg could not be detected in HepCHLine-4 cultivate supernants,either.
【Conclusion】
1 We establish a new hybrid cell line HepCHLine-4 successfully.HepCHLine-4 inherits the characteristics of both HepG2 and human primary hepatocyte,and can be used for establishing an in vitro HBV infection cell model.
2 The hybrid cell line HepCHLine-4 did not contain HBV genome prior to HBV infection experiment,so it can be a useful tool to study the process of HBV infection.
SectionⅡInvestigate the Susceptibility of Hybrid Cell Line HepCHLine-4 to Serum-derived HBV Virions
【Objective】
In order to investigate the possibility of hybrid cell line HepCHLine-4 served as an in vitro HBV infection cell culture system,HepCHLine-4 cells were co-incubated with serum-derived HBV virions,and the susceptibility of HepCHLine-4 to HBV infection was evaluated.
【Methods】
1.Co-incubated HepCHLine-4 cells and HepG2 cells with serum-derived HBV virions respectively,here HepG2 cells were set as control.
2.We analyzed intracellular HBV DNA in infected cells and HBV DNA in cultivate supernants of infected cells.HBV genome was amplified by nested PCR using the universal primers for the outer primers,followed by a mixture containing B and C type-specific inner primers.
3.We analyzed intracellular HBV cccDNA in infected cells by nested PCR after cell genome digested with mung bean nuclease.
4.We detected the capacity of HepCHLine-4 cells expressed HBV specific antigens post infection,HBcAg in infected cells were analyzed by indirect immunofluorescence staining.HBsAg and HBeAg in the supernatants of infected cells were identified by electrochemiluminescence.
5.Examination of HBV-specific particles that infected HepCHLine-4 cells released into the supernants by electron microscopy.
【Results】
1.HBV DNA was detected both in HepCHLine-4 cells and in the culture media consistently between days 6 and 15 post infection,whereas could not be detected in infected HepG2 cell or its culture media.
2.HBV cccDNA was detected consistently between days 5 and 10 p.i.of HepCHLine-4 cells,whereas could not be detected in infected HepG2 cells.
3.(1) The core antigen was detected throughout the cytoplasm of HepCHLine-4 cells between days 1 to 30 p.i.,and in the nucleus of HepCHLine-4 cells post infection between days 2 to 30 p.i.;whereas could not be detected within HBV-infected HepG2 cells.(2) HepCHLine-4 cells consistently secreted HBsAg between days 6 and 15 p.i.,and HBsAg in culture media was from 3.850 to 89.590 IU/mL(≥1 was considered positive).HepCHLine-4 cells consistently secreted HBeAg between days 8 and 17 p.i.,and HBeAg in culture media was from 1.000 to 1.690 S/Co(≥1 was considered positive).HBsAg and HBeAg could not be detected in culture media of infected HepG2 cells.
4.Culture media of the 6~(th) day post infection were collected and subjected to ultracentrifugation.The pelleted viral particles were directly examined by electron microscopy after negative staining with 2%phosphotungstic acid.22-nm-diameter spherical and 22-nm-long filamentous forms HBsAg particles as well as few 42-nm-diameter double-shelled Dane-like particles were detected.However,no HBV-associated particles were found when similar samples were examined from culture media of the control,HepG2 cells.
【Conclusion】
The results indicated that the hybrid cell line HepCHLine-4 was susceptible to serum-derived HBV virions.HepCHLine-4 cells post infection were found to be able to initiate viral DNA replication and the production of replicative intermediate and HBV-specific protein.Furthermore,HBV-specific viral particles were produced and released into culture media.
Till now,HepCHLine-4 cell has experienced more than one year,and the cell strain can be resuscitated after being freezed.HepCHLine-4 was morphologically unchanged after the 50th subculture,and maintained its susceptibility to HBV.
【In conclusion】
All these results combined indicate that the hybrid cell line HepCHLine-4 inherits the high susceptibility to HBV from human hepatocytes;on the other hand,it inherits the immortality of HepG2 cells,overcoming the deficiency of primary human hepatocytes as a HBV infection system.HepCHLine-4 cell line is susceptible to HBV and can initiate viral replication,produce HBV-specific antigens and HBV-specific progeny virions after HBV infection.In conclusion,HepCHLine-4 cell line is a suitable cell model for studying the early events of HBV infection and analyzing the in vivo process of viral infection and viral life cycle in human hepatocytes.
乙型肝炎病毒(hepatitis B virus,HBV)是嗜肝DNA病毒属的一员。HBV可以引起肝脏的急、慢性感染,HBV的慢性感染是引起严重肝脏疾病(比如:肝硬化、肝细胞肝癌)的主要病因。据世界卫生组织报道,全球20多亿人曾感染乙肝病毒,其中3.5亿以上为慢性乙型肝炎病毒感染者。慢性乙型肝炎不易彻底治愈,并且具有发展成肝硬化、肝癌的高度危险性。每年全世界范围内约有一百万人死于慢性乙型肝炎感染所致的肝衰竭、肝硬化和肝细胞肝癌。我国约有超过50%的居民曾被乙肝病毒感染过,其中8%～15%的人成为慢性感染者。
自从1965年Blumberg等发现乙型肝炎病毒表面抗原,到1997年乙肝病毒全基因组的克隆和测序完成,人们对HBV的基因组成、抗原结构、生物学特性及其生命周期的认识迅速发展。但是由于嗜肝DNA病毒属的感染具有严格的宿主特异性和组织特异性,限制了适合于研究人HBV感染的体外细胞模型的建立。
目前,用于体外研究HBV的细胞模型主要是人原代肝细胞和肝癌细胞来源的细胞系。人原代肝细胞用于HBV感染研究有其特有的优势:人原代肝细胞保留了分化肝细胞的重要细胞学特性,允许HBV的自然穿入和完整复制。然而,其缺陷在于在体外培养几天后,原代肝细胞开始出现肝细胞特殊形态的消失,并且失去了对HBV的感染和复制能力。此外,人原代肝细胞必须由新鲜肝脏组织获得,来源有限,并且其对病毒的摄取能力还受到细胞生长状态的影响。人原代肝细胞培养因为存在上述困难而限制了其应用,对HBV的研究更多的依赖于转染了HBV DNA克隆的分化良好的肝癌细胞系。其中最常用的是HepG2.2.15细胞系,HepG2.2.15细胞稳定转染了完整的HBV基因组,表达HBV RNA、病毒蛋白并能产生病毒样颗粒。由HepG2.2.15细胞分泌的病毒颗粒已经被证实可以感染黑猩猩。目前我们对于HBV复制和病毒基因组组成的许多重要的知识都是通过对转染了HBV DNA的肝癌细胞系的研究获得的。但是,由于HBV基因组是通过转染而非自然感染的方式进入到细胞中的,这些转染了HBV DNA的肝癌细胞系不能用于对HBV感染宿主细胞的早期感染过程的研究,包括病毒吸附,穿入和脱壳等过程。
由于缺乏有效的体外病毒感染和复制模型系统,HBV感染肝脏的早期机制研究进展较为缓慢,从而在一定程度上影响了乙型肝炎及其相关肝脏疾病的基础和临床研究。本研究旨在建立一个适合于HBV自然感染,并能有效进行病毒复制的细胞模型,为HBV感染宿丰细胞的早期机制,以及病毒侵入人肝细胞后完整的复制过程的研究提供一个有用的研究工具。
具体实验内容如下:
第一部分杂交细胞株HepCHLine-4的建立
【目的】
建立人原代肝细胞与HepG2的杂交细胞,以作为后续实验研究的基础。
【方法】
1建立人原代肝细胞与HepG2的杂交细胞。
1.1利用化学诱变剂诱导HepG2细胞产生次黄嘌呤鸟嘌呤磷酸核糖转移酶(HGPRT)基因突变。
1.2人原代肝细胞的分离与培养,台盼蓝染色排除法确定肝细胞存活率。
1.3利用细胞融合技术,将筛选出的HGPRT缺陷的HepG2细胞(HGPRTˉHepG2细胞)与人原代肝细胞进行融合,经HAT培养基筛选出异核体杂交细胞,进而用有限稀释法进行单克隆。
1.4利用胰蛋白酶G显带技术对所得细胞进行核型分析。
1.5绘制杂交细胞生长曲线。
2对杂交细胞是否携带HBV基因进行检测。
因为本研究中融合成功的杂交细胞是用慢性乙型肝炎携带者的肝细胞与HepG2细胞融合所得,所以在进行HBV感染实验之前我们对杂交细胞是否携带HBV基因进行检测。
2.1合成HBV型特异性引物,用巢式PCR检测杂交细胞内及培养上清中是否存在HBVDNA。
2.2提取杂交细胞基因组DNA,绿豆核酸酶消化后,巢式PCR检测杂交细胞内是否存在HBV的复制中间产物HBV cccDNA。
2.3杂交细胞是否表达HBV特异性蛋白抗原:间接免疫荧光检测杂交细胞内是否有HBcAg的表达;电化学发光法检测杂交细胞培养上清中的HBsAg和HBeAg。
【结果】
1成功建立人原代肝细胞与HepG2的杂交细胞。
1.1经EMS诱导突变得到的HGPRTˉHepG2细胞能耐受6-MP但不能在HAT选择培养基中生长,说明HGPRTˉHepG2细胞中次黄嘌呤核苷酸焦磷酸化酶(IPP)完全缺失。
1.2人原代肝细胞经台盼蓝染色排除法确定存活率≥90%。
1.3将HGPRTˉHepG2细胞和分离得到的人原代肝细胞融合后,经HAT选择培养基筛选和单克隆后,得到一株杂交细胞克隆,命名为HepCHLine-4。HepCHLine-4细胞在形态学上与HGPRTˉHepG2细胞相似,能传代培养,传代一次约需3～5天,经冻存复苏后可继续生长。
1.4染色体核型分析结果显示野生型HepG2细胞、HGPRTˉHepG2细胞、HepCHLine-4细胞染色体众数分别为51条、53条和99条,证实HepCHLine-4是一杂交细胞株,为HGPRTˉHepG2细胞(53条染色体)和人原代肝细胞(46条染色体)的杂合体,包含所有来自HGPRTˉHepG2细胞和人原代肝细胞的基因,兼有这两种细胞的遗传特性。
1.5绘制杂交细胞生长曲线,结果显示杂交细胞HepCHLine-4符合传代细胞生长过程特点。
2杂交细胞自身不携带HBV基因。
2.1杂交细胞内及培养上清中均未检测到HBV DNA。
2.2杂交细胞内不存在HBV的复制中间产物HBV cccDNA。
2.3杂交细胞不能表达HBV特异性蛋白抗原:杂交细胞内没有检测到HBcAg的表达;杂交细胞培养上清中没有检测到HBsAg和HBeAg。
【结论】
1.成功建立了一个兼具有HepG2和人原代肝细胞遗传特性的杂交细胞株HepCHLine-4,可以进一步用来研究建立适宜于HBV感染的细胞模型。
2.杂交细胞HepCHLine-4虽然是由慢性乙型肝炎携带者肝细胞与HepG2细胞融合所得,但是并不携带HBV基因及HBV的复制中间产物,故可以用于HBV感染的实验研究。
第二部分评价杂交细胞株HepCHLine-4对HBV的自然感染能力
【目的】
用慢性乙型肝炎患者血清中的病毒颗粒自然感染杂交细胞,评价杂交细胞对HBV的自然感染能力,探讨杂交细胞株HepCHLine-4用作HBV感染细胞模型的可能性。
【方法】
1.用含HBV DNA的慢性乙型肝炎患者血清分别与杂交细胞HepCHLine-4和HepG2细胞共同孵育,感染细胞,HepG2细胞作为对照细胞。
2.用HBV型特异性引物,巢式PCR检测感染后细胞内HBV DNA合成及分泌情况。
3.提取感染后细胞基因组DNA,绿豆核酸酶消化后,巢式PCR检测感染后细胞内有无HBV复制中间产物HBV cccDNA。
4.感染后杂交细胞中HBV特异性蛋白合成及分泌能力的检测:间接免疫荧光检测感染后细胞内是否有HBcAg的表达;电化学发光法检测感染后细胞培养上清中的HBsAg和HBeAg。
5.电镜检查感染后细胞能否向培养上清中分泌HBV病毒颗粒。
【结果】
1.HBV感染后的第6天到第15天,HepCHLine-4细胞内和培养上清中均能检测到HBV DNA;而感染后的HepG2细胞内及培养上清中均未检测到HBV DNA。
2.HBV感染后的第5天到第10天,HepCHLine-4细胞内均可以检测到HBV的复制中间产物HBV cccDNA的存在;而感染后的HepG2细胞内未检测到HBVcccDNA。
3.(1)HBV感染后的第1天到第30天(30天之后没有再进行检测),HepCHLine-4细胞胞浆内HBcAg始终是阳性表达,间接免疫荧光染色呈胞浆弥漫性着色;细胞核内HBcAg的阳性颗粒从感染后第2天到第30天都可以观察到,阳性率约15%,间接免疫荧光染色后表现为细胞核部分着色;而感染后的HepG2细胞内未检测到HBcAg的表达。(2)HBV感染后的第6天到第15天,HepCHLine-4细胞持续分泌HBsAg到培养上清中,培养上清中检测出的HBsAg的滴度最低值为3.850IU/mL,最高值为89.590IU/mL(≥1为阳性):HBV感染后的第8天到第17天,HepCHLine-4细胞持续分泌HBeAg到培养上清中,培养上清中检测出的HBeAg的滴度最低值为1.000S/Co,最高值为1.690S/Co(≥1为阳性);而感染后的HepG2细胞培养上清中未检测到HBsAg和HBeAg。
4.电镜结果:收集HBV感染后第6天的HepCHLine-4细胞培养上清,超速离心后,沉淀用2%磷钨酸负染,JEM-1200EX透射电镜观察。电镜下可以见到少量直径42nm的双层壳状Dane颗粒和较多的直径22nm的小球型颗粒及丝状颗粒。而感染后第6天的HepG2细胞培养上清超速离心后的沉淀电镜检查结果为阴性。
【结论】
杂交细胞HepCHLine-4可以被慢性乙型肝炎患者血清中的HBV病毒自然感染,感染后病毒可以侵入杂交细胞并在杂交细胞内进行病毒基因的复制,形成复制中间产物HBV cccDNA;并且进一步进行病毒基因的表达,合成HBV特异性蛋白HBsAg、HBeAg和HBcAg;HBV感染后杂交细胞能合成子代病毒颗粒并分泌出细胞。
到目前为止,经过了一年多的冻存复苏和传代培养,杂交细胞HepCHLine-4已经传代50代次以上,细胞形态和生长特性没有明显改变,并且保持其对HBV的易感性。
总结
本研究结果证实,我们建立的杂交细胞株HepCHLine-4兼具有HepG2和人原代肝细胞的遗传特性。它一方面继承了人原代肝细胞对HBV的易感性,另一方面,继承了HepG2细胞能在体外传代培养的特性,克服了人原代肝细胞作为HBV感染细胞模型的缺陷。HepCHLine-4对HBV自然感染易感,并能在感染后启动有效的病毒复制,形成复制中间产物HBV cccDNA,合成HBV特异性抗原并能合成子代病毒颗粒分泌出细胞。因此,杂交细胞株HepCHLine-4可以作为适宜于HBV自然感染的细胞模型,进行HBV感染宿主细胞的早期机制,包括病毒黏附、穿入、脱壳等早期感染过程的研究和病毒侵入人肝细胞后完整的复制过程的研究。
Background
Hepatitis B virus(HBV) is a member of the Hepadnavirus family.HBV causes transient and chronic infections of the liver.Chronic infection with HBV is a leading cause of severe liver diseases,such as cirrhosis and hepatocellular carcinoma.More than 2 billion people alive today have been infected by HBV,and more than 350 million people are chronically infected.Patients chronically infected with HBV run the risk of developing cirrhosis and hepatocellular carcinoma in later life.Worldwide deaths from chronic liver diseases and hepatocellular carcinoma caused by HBV infection approximately 1 million each year.In China,more than 50%of the population has been infected by HBV,and 8%to 15%of them become chronically infected.
Since HBV surface antigen was found by Blumberg in 1965 to the complete cloning and sequencing of HBV DNA genome in 1997,our knowledge on genetic constitution,antigenic structure,biology feature and life cycle of HBV have been riched.But Hepadnavirus infection is of strictly host specific and tissue specific, which hampers the establishment of suitable cell model for studying HBV infection.
Currently,there are two main cell types used for the study of HBV in vitro, primary human hepatocytes and hepatoma-derived cell lines.Primary human hepatocytes for infection studies have the advantage that they retain important cellular characteristics of differentiated hepatocytes allowing natural penetration,and the full replication of HBV.However,difficulties arise with this system because within several days in culture the cells begin to dedifferentiate and lose the capability to support HBV infection and replication.In addition,these cells must be obtained from a fresh liver,and the response of viral uptake varies greatly according to condition of harvested cells.Because of the difficulties with primary cell cultures,HBV research has relied upon the development of well-differentiated human hepatoma cell lines transfected with cloned HBV DNA.One of these cell lines,HepG2.2.15,is stably transfected with the complete HBV genome,expressing HBV RNA,viral proteins, and secreting virus-like particles.The virus secreted by HepG2.2.15 has demonstrated infectivity in chimpanzees.Many important details of HBV replication and genomic organization have been discovered through the use of transfected hepatoma cell lines. However,because the genome is introduced into cells by transfection rather than infection,these cell lines cannot be used to study HBV-host cell infection processes such as viral attachment,penetration,and uncoating.
Due to deficiency of an in vitro system for productive viral infection and replication,little has been known about the mechanisms of the early stages in HBV infection.Thereby,basic and clinical research on hepatitis B and chronic liver diseases correlated with HBV infection were hampered to some extent.Here,we have attempted to establish an in vitro infection cell culture system,which is susceptible to HBV infection and suitable for replication ensued.It could be a useful tool for investigating the mechanisms of the early stages in HBV infection,and analyzing the in vivo process of viral infection and viral life cycle in human hepatocytes.
SectionⅠEstablishing a Hybrid Cell Line HepCHLine-4
【Objective】
To establish a hybrid cell line susceptible for HBV infection by fused HepG2 cells with primary human hepatocytes.
【Methods】
1 To establish a hybrid cell line by fused HepG2 cells with primary human hepatocytes.
1.1 HepG2 cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HGPRT),were induced by EMS.
1.2 Isolation and culture of human primary hepatocytes.Hepatocytes viability was defined as the ability of cells to exclude trypan blue dyeing.
1.3 HGPRT~- HepG2 cells were fused with human primary hepatocytes.After being selected with HAT medium and limiting dilution assayed,the hybrid cell was monocloned.
1.4 The modal chromosome numbers in hybrid cell was counted by the trypsin G-banding method.
2 Since the primary hepatocytes we used for cell fusion was come from a patient chronically infected with HBV,it is necessary to analyze whether HepCHLine-4 contains HBV genome prior to HBV infection experiment.
2.1 We analyzed intracellular HBV DNA and HBV DNA in cultivate supernants. HBV genome was amplified by nested PCR using the universal primers for the outer primers,followed by a mixture containing B and C type-specific inner primers.
2.2 We analyzed intracellular HBV cccDNA by nested PCR after HepCHLine-4 cell genome digested with mung bean nuclease.
2.3 We detected whether HepCHLine-4 cells expressed HBV specific antigens. HBcAg in HepCHLine-4 cell was analyzed by indirect immunofluorescence staining.HBsAg and HBeAg in the supernatants were identified by electrochemiluminescence.
【Results】
1 We establish a hybrid cell line successfully by fused HepG2 cells with primary human hepatocytes.
1.1 HepG2 cells deficient in HGPRT(HGPRT~- HepG2) were induced by EMS. HGPRT~- HepG2 cells were resistant to 6-MP but could not grow in HAT medium, indicating a complete defect of inosinic acid pyrophosphorylase(IPP).
1.2 Human primary hepatocytes viability was defined≧ 90%.
1.3 After cell fusion and being selected with HAT medium and limiting dilution assayed,we established a bybrid cell line,named HepCHLine-4.HepCHLine-4 was similar to HGPRT~- HepG2 cells morphologically,and can be subcultured in vitro.HepCHLine-4 cell line can be resuscitated after being freezed.
1.4 The Karyotype analysis results showed that the modal chromosome numbers of wild-type HepG2,HGPRT~- HepG2 and HepCHLine-4 were 51,53 and 99, respectively,indicating that the HepCHLine-4 was a hybrid cell line between HGPRT~- HepG2 cells(53 chromosomes) and human hepatocytes(46 chromosomes),and contained all genomic factors from both human hepatocytes and HepG2 cells.
2 The hybrid cell line doesn't contain HBV genome prior to HBV infection experiment.
2.1 HBV DNA could not be detected in HepCHLine-4 cells or in the cultivate supernants.
2.2 HBV cccDNA,the replicative intermediate of HBV,could not be detected in HepCHLine-4 cells.
2.3 HepCHLine-4 cells could not express HBV specific antigens prior to HBV infection.HBcAg could not be detected in HepCHLine-4 cells.HBsAg and HBeAg could not be detected in HepCHLine-4 cultivate supernants,either.
【Conclusion】
1 We establish a new hybrid cell line HepCHLine-4 successfully.HepCHLine-4 inherits the characteristics of both HepG2 and human primary hepatocyte,and can be used for establishing an in vitro HBV infection cell model.
2 The hybrid cell line HepCHLine-4 did not contain HBV genome prior to HBV infection experiment,so it can be a useful tool to study the process of HBV infection.
SectionⅡInvestigate the Susceptibility of Hybrid Cell Line HepCHLine-4 to Serum-derived HBV Virions
【Objective】
In order to investigate the possibility of hybrid cell line HepCHLine-4 served as an in vitro HBV infection cell culture system,HepCHLine-4 cells were co-incubated with serum-derived HBV virions,and the susceptibility of HepCHLine-4 to HBV infection was evaluated.
【Methods】
1.Co-incubated HepCHLine-4 cells and HepG2 cells with serum-derived HBV virions respectively,here HepG2 cells were set as control.
2.We analyzed intracellular HBV DNA in infected cells and HBV DNA in cultivate supernants of infected cells.HBV genome was amplified by nested PCR using the universal primers for the outer primers,followed by a mixture containing B and C type-specific inner primers.
3.We analyzed intracellular HBV cccDNA in infected cells by nested PCR after cell genome digested with mung bean nuclease.
4.We detected the capacity of HepCHLine-4 cells expressed HBV specific antigens post infection,HBcAg in infected cells were analyzed by indirect immunofluorescence staining.HBsAg and HBeAg in the supernatants of infected cells were identified by electrochemiluminescence.
5.Examination of HBV-specific particles that infected HepCHLine-4 cells released into the supernants by electron microscopy.
【Results】
1.HBV DNA was detected both in HepCHLine-4 cells and in the culture media consistently between days 6 and 15 post infection,whereas could not be detected in infected HepG2 cell or its culture media.
2.HBV cccDNA was detected consistently between days 5 and 10 p.i.of HepCHLine-4 cells,whereas could not be detected in infected HepG2 cells.
3.(1) The core antigen was detected throughout the cytoplasm of HepCHLine-4 cells between days 1 to 30 p.i.,and in the nucleus of HepCHLine-4 cells post infection between days 2 to 30 p.i.;whereas could not be detected within HBV-infected HepG2 cells.(2) HepCHLine-4 cells consistently secreted HBsAg between days 6 and 15 p.i.,and HBsAg in culture media was from 3.850 to 89.590 IU/mL(≥1 was considered positive).HepCHLine-4 cells consistently secreted HBeAg between days 8 and 17 p.i.,and HBeAg in culture media was from 1.000 to 1.690 S/Co(≥1 was considered positive).HBsAg and HBeAg could not be detected in culture media of infected HepG2 cells.
4.Culture media of the 6~(th) day post infection were collected and subjected to ultracentrifugation.The pelleted viral particles were directly examined by electron microscopy after negative staining with 2%phosphotungstic acid.22-nm-diameter spherical and 22-nm-long filamentous forms HBsAg particles as well as few 42-nm-diameter double-shelled Dane-like particles were detected.However,no HBV-associated particles were found when similar samples were examined from culture media of the control,HepG2 cells.
【Conclusion】
The results indicated that the hybrid cell line HepCHLine-4 was susceptible to serum-derived HBV virions.HepCHLine-4 cells post infection were found to be able to initiate viral DNA replication and the production of replicative intermediate and HBV-specific protein.Furthermore,HBV-specific viral particles were produced and released into culture media.
Till now,HepCHLine-4 cell has experienced more than one year,and the cell strain can be resuscitated after being freezed.HepCHLine-4 was morphologically unchanged after the 50th subculture,and maintained its susceptibility to HBV.
【In conclusion】
All these results combined indicate that the hybrid cell line HepCHLine-4 inherits the high susceptibility to HBV from human hepatocytes;on the other hand,it inherits the immortality of HepG2 cells,overcoming the deficiency of primary human hepatocytes as a HBV infection system.HepCHLine-4 cell line is susceptible to HBV and can initiate viral replication,produce HBV-specific antigens and HBV-specific progeny virions after HBV infection.In conclusion,HepCHLine-4 cell line is a suitable cell model for studying the early events of HBV infection and analyzing the in vivo process of viral infection and viral life cycle in human hepatocytes.
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