【作者】 王刚；

【导师】 马立宪；

【作者基本信息】 山东大学 ， 内科学， 2005， 博士

【摘要】 研究背景和目的 慢性乙型肝炎是全球性的重大健康问题。约15%-20%存在病毒活跃复制的慢乙肝患者,在5年内发生肝硬化;慢性乙肝患者罹患原发性肝细胞癌(Hepatocellular carcinoma,HCC)的相对危险性至少增加100-300倍。乙型肝炎的病原体—乙型肝炎病毒(HBV)属于嗜肝DNA病毒(Hepadnaviridae),具有较严格的宿主特异性和组织嗜性,其仅感染人类和某些高级灵长类动物,而肝细胞是目前唯一证实的HBV体内复制场所。由于缺乏理想的HBV体外感染模型和小动物模型,因此限制了HBV相关的分子生物学、致病机理及治疗药物等多方面的研究。目前许多嗜肝DNA病毒的研究成果均来自于鸭乙型肝炎病毒(DHBV)及土拨鼠肝炎病毒(WHV),由于HBV与上述两种病毒间仍存在较大的差异,所以有些已知的研究结论可能并不适用于HBV。因此迫切需要建立支持HBV自然感染的实验模型。 HepG2等肝肿瘤细胞株经常用于HBV体外实验,这类细胞可以连续传代,且一定程度上具有肝细胞的生物学特性,可能是潜在的比较理想的HBV细胞候选模型。然而由于某些未知因素限制,这类细胞无法自然感染HBV。但通过人工转染病毒基因,不仅可以实现HBV表达、复制,亦可以实现DHBV的复制。表明HepG2可以提供嗜肝DNA病毒复制所需的条件,HBV感染HepG2细胞可能仅仅受限于病毒侵入的早期过程。然而目前缺乏相关研究,对于HBV早期感染过程还知之甚少,也不清楚究竟是哪一个或哪一些环节限制了HBV自然感染HepG2细胞。更多还原

【Abstract】 BACKGROUND AND OBJECTIVE OF RESEARCHChronic hepatitis B (CHB) is a serious problem of common health worldwide. Among patients with active viral replication, cirrhosis will develop in 15 to 20 percent within five years. Patients with CHB have a significantly increased relative risk of hepatocellular carcinoma. Pathogen of CHB is hepatitis B virus (HBV), which belongs to hepadnaviruses. HBV has narrow host range and strict tissue tropism. It only infect human and higher primates such as chimpanzee. Hepatocytes are the only confirmed site of replication for HBV in vivo. Because of lack of ideal cell model and little animal model, the research on molecular biology, pathogenesis and therapy of HBV have been hampered. So far most of research conclusions about hepadnaviruses come from study on duck hepatitis B virus (DHBV) and woodchuck hepatitis virus (WHV). Since there are great differences between HBV and DHBV as well as WHV, some known conclusions maybe do not hold true for HBV. Therefore, it is urgent to establish ideal cell model that support HBV natural infection.The hepatoma cell lines, such as HepG2, are often used in experiments about HBV in vitro. These cell lines can successively subculture, and have biology properties of hepatocyte to a certain extent. They are maybe the potential ideal modelfor HBV infection. However, owing to some unknown restrictive factors, they are nonpermissive for HBV natural infection. As HepG2 can support both HBV and DHBV replication after transfection of viral DNA, it is likely that the block in HBV infection is attributed to an early step of viral infection cycle. But related studies are absent, resulting that early events of HBV infection are ill-defined. We also do not know which factor limits HepG2 supporting HBV infection.In this study, the aims are to explore the mode of HBV adsorption and penetration with HepG2 cells, to investigate the factors which affect HBV adsorption and penetration, to primarily explore the cellular receptor for HBV, to further study the viral location following penetration and to illuminate the limiting factors of HBV natural infection infinite cell line.MATERIALS AND METHODSThe first part1. HepG2 cells were grown in 6-well plates at 5×106 cells/well in Dulbecco’s modified Eagle’s medium (DMEM)2. HBV virions came from serum of asymptom carrier with a high titer of HBV (6.23 × 109copies/ml determined by fluorescence quantitative-polymerase chain reaction, FQ-PCR), then sterilized by sieving and stored at -70 ℃ prior to use.3. HBV-adsorption assay0.1ml of serum containing HBV virion was diluted by 0.3ml DMEM, then added to plates and incubated with HepG2 cells at 4℃ for 2h, afterwards one of the 6-well plate was washed three times with cold PBS, and subsequently trypsinized with 0.125% trypsin, trypsinization solution and HepG2 cells were collected for the further assay. Above steps were operated on ice. Another 6-well plate was placed at 37℃ following incubation at 4℃, 4h later, trypsinized ditto. HBV adsorbed HeLa cells as control.4. HBV-adsorption interference assay4.1 Pretreatment of cells with trypsinPrior to inoculated with HBV, HepG2 cells were trypsinized with 0.125% trypsin at 37 ℃ for 3min followed by the careful addition of fresh culture medium to inactivate trypsin. Subsequent steps are idem (details seen HBV-adsorption assay).4.2 Pretreatment of cells with heparinaseⅢPrior to inoculated with HBV, HepG2 cells were treated with 1 unit/ml heparinaselll in DMEM at 37 ℃ for 1h. Treated cells were then washed twice with common DMEM, subsequently operated ditto.5. Preparation of nucleic acids from both cell and trypsinization solution.6. HBV DNA, HBV covalently closed circular DNA (cccDNA) were detected by PCR, FQ-PCR and selective PCR, respectively. Distribution of HBV surface antigen (HBsAg) within HepG2 cells was examined by Indirect Immunofluorescence (IIF).The second part1. Cells culture and viral preparation were the same as the first part.2. In order to acquire more mitotic cells, HepG2 cells were synchronized at low temperature, then HepG2 cells were incubated with HBV at 37 ℃ for 2h, Subsequently, culture supernate and HepG2 cells were collected at 4h、 8h、 24h、 48h and 96h post-inoculation, respectively. HBV infected HeLa cells as negative control.3. Parts of collected cells were partitioned into nuclear and cytoplasmic components. DNA were extracted from supernate and cellular components by routine methods.4. HBV DNA were detected by the same methods as the first part. Location and distribution of HBV core antigen (HBcAg) within HepG2 cells were examined by IIF too. Viral antigens in culture supernate were detected by enzyme-linked immunosorbent assay (ELISA).RESULTSThe first part1. HBV adsorption HepG2 cells at temperature of 4℃Cells surface-bound virus could be removed by trypsinization. HBV DNA was positive in trypsinization solution but negative in HepG2 cells by PCR. HBsAg within HepG2 cells was also negative by IIF. This results suggested that HBV was merely able to bind to HepG2 cells surface at temperature of 4℃, it did not occurred that HBV entered into cytoplasm.2. HBV adsorption HepG2 cells at temperature of 37℃HBV DNA was positive both in trypsinization solution and HepG2 cells by PCR assay. When HBsAg was detected by IIF, green fluorescence signal could be seen in both cytomembrane and cytoplasm. This results suggested HBV could penetrate into cytoplasm of HepG2 cells at 37℃.As both DNA and antigen of HBV were negative within Hela cells under the same conditions, it was concluded that HBV adsorbed specially HepG2 cells.3. HBV-adsorption kineticsIn this experiment, we used a constant number of HepG2 cells. Initially, with increasing of inoculated HBV titer the amounts of HBV DNA within trypsinization solution also augmented. When the concentration of inoculated HBV exceeded 4 X 108copies/ml, the amounts of HBV DNA within trypsinization solution leveled off and did not increased any more. This result confirmed that the HBV-cell binding reaction have saturation. Comparison of bound and internalized virus showed that only a proportion, estimated to be 27%, of cell-bound virus was internalized. Further comparison with the number of cells concluded that approximately 5 virus particles per cell were internalized.4. HBV-adsorption interference assay 4.1 Pretreatment of cells with trypsinHBV DNA was negative both in trypsinization solution and HepG2 cells by PCRassay. This test showed that HBV could not adsorb and penetrate HepG2 cells when the extracellular domains of cytomembrane proteins were cleaved by trypsin. 4.2 Pretreatment of cells with heparinaseⅢHBV DNA was positive both in trypsinization solution and HepG2 cells by PCR assay. Brightness of electrophoresis bands had not distinct difference by comparison with positive control. This result indicated that glycosaminoglycans on cellular surface had not significant contribution to HBV adsorption as well as internalization. The second part1. Synchronization at low temperatureHepG2 cells were synchronized at low temperature. 85% of cells lay metaphase at 2h post-inoculation. Most of cells had ended mitotic phase at 4h pot-inoculation. Cells at metaphase were about 10 percent of all cells.2. HBV-infection assayAfter incubation at 37 ℃, HBV DNA could be detected in HepG2 cells at 4h-24h., however, viral DNA became weakly positive and negative at 48h and 96h, respectively. Antigens and DNA of HBV in culture supernate were all along negative at various times after incubation at 37 ℃. Initially, internalized HBV DNA was detected mainly in the cytoplasm, subsequently Cytoplasmic fraction showed reduced levels of viral DNA and became negative at 24h post-incubation. In contrast, HBV was congregated to nucleus. Viral DNA within the nucleus increased from 4h to 24h after incubation, but became weakly positive at 48h, and disappeared at 96h. HBV DNA was detected both in nucleoplasm and nuclear membrane at 8h post-inoculation. Using method of IIF, HBc could be also detected in both nucleoplasm and nuclear membrane at 8h post-inoculation. The results showed that viral genome and HBc had tropism for nucleus after HBV penetrated into cells, however, since HBV could not achieve replication in HepG2 cells, HBV within cells was subjected to degrade and disappeared finally with time prolonging.3. Detecting cccDNAThrough method of selective PCR, HBV cccDNA was all along negative withinHepG2 cells which were collected from every phase. It means that HBV could not form intermediate of replication. This result further confirmed that HBV could not establish productive infection in HepG2 cells.CONCLUSIONS1. This study demonstrated that the HBV-HepG2 cell binding reaction satisfied criteria of receptor-ligand interaction, and this binding reaction had saturation.2. HBV probably infected HepG2 cells by co-receptors mode. This process involved multi-proteins interaction between host cell and virus.3. Proteins of cytomembrane is the main effective molecule for HBV adsorbing HegG2 cells, glycosaminoglycans on cellular surface had not significant contribution to HBV adsorption as well as penetration.4. HBV was nucleus-tropic after penetrating into cell. There were both nucleic acid and proteins of HBV in nucleus of HepG2 cells.5. The failure of uncoating viral genome maybe was the main limiting factor that led to HepG2 cells retractile to HBV replicative infection.更多还原