Expression Of HBV1.3-Fold Genome Plasmids In An SV40T-Immortalized Mouse Hepatic Cell Line

BackgroundHepatitis 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. More than2billion people alive today have been infected by HBV, and more than350million 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 approximately1million each year. In China, more than50%of the population has been infected by HBV, and8%to15%of them become chronically infected.The goal of CHB treatment is to achieve the continuous suppression of hepatitis B virus (HBV)’s replication and the remission of liver disease. Treatment for hepatitis B depends on several factors, such as stage of disease, the presence or absence of the "e" antigen, the potential for drug resistance and subsequent inability to use a medicine, particularly in the final stages of chronic disease the liver. Therefore, it is very important to evaluate these factors at the time of choosing the type and duration of treatment. Interferon alpha-2a and interferon alfa-2b (IFN2a and2b) has been used for many years as the first choice of treatment for low levels of HBV DNA and high levels of alanine aminotransferase (ALT). The goal of treatment is to activate an immune response in terms of HBeAg seroconversion. This form of treatment is the first option to change the immune system, aims to obtain the elimination or remission. However, the treatment is very expensive, and also has many side effects, such as anaemia, a greater decrease in hemoglobin, vomiting, cold sweats and nausea. Only about one out of three patients received the IFN therapy. Furthermore, nucleoside analogues can not completely eliminate the virus, and may lead to the mutation of virus. So the development of new antiviral treatment remains a major research task. And the new suitable HBV-infected cell animal models are urgently required to evaluate new treatment strategies.Currently, HepG2.2.15cell line is the most commonly used HBV cell model in vitro studies. HepG2.2.15cell line is capable of supporting HBV replication and secretion of infectious virus particles and is recognized stable cell lines of HBV gene expression. But as an in vitro HBV infection, especially as the biological characteristics of the antiviral drug screening and drug-resistant mutants cell model, HepG2.2.15cell line has certain limitations because of the viral gene integrated into the host cell chromosome with relative fixed number of copies, and viral gene has low expression levels. So it Is still an urgent need to establish an in vitro cell culture or experimental animal models to help us understand the HBV infection of the virus-host interactions, determine the pathogenicity of the virus mutants, test new antiviral drugs and choice of treatment of chronic infection.It is the common method to use viral gene transduction to establish a cell lines. It is the most common method of cell immortalization to transfect the SV40early transcribed region of genes into cells. This mathod is applicable to variety of human cell types. Through in-depth study of the gene mediated by the SV40T immortalized cell lines, most studies show that the import of the SV40T gene in addition to increase the growth rate of transformed cells able to retain its original cell differentiated phenotype, rather than the non-original gene expression is rare. Immortalized cell may reflect the biological characteristics of the original cell at the cellular level and can be used as a model of in vitro studies. The1.3-fold overlength genome HBVDNA plasmids is the most used plasmids. It contains the complete HBV genome. Its genome is less than2.0-fold plasmids, the efficiency of replication and expression is higher than1.2and1.1-fold plasmids and include HBV5’end of the Enh Ⅰ, Enh Ⅱ, replication origin (DR1, DR2) pregenomic transcription start sites, x and pre-C promoter, the x open reading frame, etc.Therefore, this study was to establish a new immortalized mouse hepatic cell line induced by SV40T-antigen (SV40T) gene, and to investigate the expression of HBV1.3-fold overlength genome HBVDNA plasmids (pHBV1.3) in the established SV40T-immortalized mouse hepatic cell line.Section IEstablishing a SV40T-immortalized Mouse Hepatic Cell Line[Objective]To establish a SV40T-immortalized Mouse Hepatic Cell Line by transfected S V40T early transcribed region of genes into the mouse hepatic cells.[Methods]1、To establish a SV40T-immortalized Mouse Hepatic Cell Line.1.1、To isolate and culture primary mouse hepatocytes.1.2、To amplificate and extract SV40T gene plasmid (pRSV-T).1.3、To transfect pRSV-T plasmid into primary mouse hepatocytes by Iipofectamine2000.2、To detect the SV40T immortalized mouse liver cell line.2.1、to detect SV40T antigen and its distribution in SV40T-transfected cells by indirect immunofluorescence assay. Primary mouse hepatic cells were treated as the negative control.2.2、The inverted phase contrast microscope and electron microscope were used to observe the morphology and ultrastructure of SV40T-transfected mouse hepatic cells.2.3、The supernatant fluids of primary and immortalized mouse hepatic cell culture supernatants were collected, respectively. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alpha-fetoprotein (AFP) were determined by the automatic biochemical analyzer (Beckman). Primary cultured mouse hepatic cells were treated as control.2.4、After total RNA extraction of primary and SV40T-transfected hepatic cells by RNA extraction kit, RT-PCR was used to determine ALB-mRNA as previously described.2.5、After total RNA extraction of primary and SV40T-transfected hepatic cells by RNA extraction kit, RT-PCR was used to determine ALB-mRNA as previously described. 2.6、Western blot was used to detect the immunoreactivity of CK-18in primary and SV40T-transfected mouse hepatic cells (22-generation). Rabbit anti-mouse cell CK-18was the primary antibody, and horseradish peroxidase-conjugated goat anti-rabbit IgG were the second antibodies.[Results]1、The anti-Amp epithelial cell-like positive clone cells were found at30days after the lipofection of mouse hepatic cells transfected by SV40T, looked as adherent monolayer and flat-shaped, and presented as the polygonal, cluster-like multi-cell arrangement.2、After SV40T transfection, SV40T antigen immunofluorescence of mouse hepatic cells was gradually increased, and could be visable at30days after transfection. Matte-like fluorescence can be seen clearly in cytoplasm, and granular-like fluorescence in nucleus.3、SV40T-immortalized mouse hepatic cells showed typical morphology and structure of hepatic cells, and many glycogen granules, mitochondrias and endoplasmic reticulum structure were clearly visible in the electron microscope. And the splitting dual-core cells reflected the in-vitro proliferation and differentiation processes of the transfected hepatic cells. Cells passaged every five days according to1:2, and passaged38generations. No change in cell morphology was found in these cells.4、Primary mouse liver cells grow slowly in the medium containing10%fetal bovine serum.They began to slowly proliferate at7days and decease at8days.But Immortalized cells began to slowly proliferate at3-4days and decease at5days.5、The levels of ALT, AST and AFP in the supernatant fluid of culture fluid were shown in Table1. No significant difference in levels of ALT, AST and AFP between mouse hepatic cell and SV40T-transfected hepatic cells (P>0.05).6、After total RNA extraction of SV40T-transfected hepatic cells (22generation) and RT-PCR, ALB-mRNA was shown in the bright bands at475bp, indicating that SV40T-immortalized mouse hepatic cells have the ability to express ALB mRNA. Mouse hepatic cells were treated as the positive control7、After protein extraction of SV40T-transfected hepatic cells (22generation), SDS-PAGE and Western blot, The target band of CK-18was positive, indicating that the SV40T-immortalized mouse hepatic cells have the immunoreactivity of CK-18. Mouse hepatic cells were treated as the positive control.[Conclusion]1、PRSV-T plasmid allows the immortalization of mouse liver cells. SV40T-immortalized mouse hepatic cell lines can be establisned.2、SV40T-immortalized mouse liver cell line not only has same cell morphology and growth characteristics as primary mouse liver cell but also can be subcultured in vitro, so can be further used to establish an appropriate cellular model of HBV infection.So far, after more than five years cryopreserved and subcultured of, SV40T-immortalized mouse liver cell line has been passed down50generations and cell morphology and growth characteristics did not change significantly.Section IIExpression of HBV1.3-fold Genome Plasmids in an SV40T-immortalized Mouse Hepatic Cell Line[Objective]Observed the exppression of pHBV1.3plasmid in SV40T-immortalized mouse hepatic cell lines and hoped to establish a new HBV cell model.[Methods]1、HBV1.3-fold over length genome plasmids (pHBV1.3) were provided by Professor Yinping Lu in Huazhong University of Science&Technology. pHBV1.3contained1.3-fold over length HBV genome (ayw subtype). According to the instructions of liposome transfection kit ljpofectamine2000, pHBV1.3plasmid was used to trasfect SV40T-immortalized cells (22th generation).2、The supernatant fluids of pHBV1.3-transfected cells were collected at different time. AXSYM automatic electrochemiluminescence immunoassay analyzer (Abbott) was used to determine the levels of HBsAg and HBeAg.3、pHBV1.3-transfected cells were seeded in24-well plates. The cells were washed with PBS for three times, and fixed with4%paraformaldehyde at4℃for20 minutes. Then, the cells were washed with PBS for3times again, and sealed up with10%goat serum (PBS diluted) for30minutes. Then fluorescence test was performed by fluorescence kit.4、At72h of transfection, DNA extraction of pHBV1.3-transfected cells was performed for Southern hybridization. The probe was the Digoxigenin-labeled3.2KB HBVDNA. Southern hybridization was fufilled according to the instructions of southern kit.5、At72h of transfection, the supernatant fluids of pHBV1.3-transfected cells were collected. JEOL transmission electronic microscopy (JEM-1200EX Electron Microscope) was used to observed and take photographs.[Results]1、The levels of HBsAg and HBeAg in the supernatant fluid after transfection of pHBV1.3were detected at24,48,72and96h. The levels of HBsAg and HBeAg were increased continuously in the supernatant fluid after transfection of pHBV1.3, and began to decrease after72h. Then, the levels of HBsAg and HBeAg decreased graduatedly.2、The expressions of HBsAg and HBcAg were observed in the pHBV1.3-transfected cells at24h after transfection, and peaked at72h through fluorescence test. HBsAg expressed mainly in cytoplasm, while the expression of HBcAg was distributed in cytoplasm and nucleus, mainly in cytoplasm.3、HBV DNA replication intermediates were also observed at72h after transfection, including rcDNA, dsDNA and ssDNA through Southern hybridization.4、Culture media of the72hours post transfected were collected and subjected to ultracentrifugation. The pelleted viral particles were directly examined by electron microscopy after negative staining with2%phosphotungstic acid.22-nm-diameter spherical and22-nm-long filamentous forms HBsAg particles as well as few42-nm-diameter double-shelled Dane-like particles were detected.[Conclusion]SV40T-immortalized mouse liver cell line can be transfected by pHBV1.3plasmid. After transfected, HBVDNA can replicate in SV40T-immortalized mouse liver cell line, such as that can formate virus replication intermediates including rcDNA, dsDNA and ssDNA and synthesis HBV-specific protein of HBsAg, HBeAg and HBcAg and progeny virus particles that can be secreted out of the cell.[In conclusion]The results indicated that PRSV-T plasmid allows the immortalization of mouse liver cells. SV40T-immortalized mouse hepatic cell lines can be establisned.SV40T-immortalized mouse liver cell line not only has same cell morphology and growth characteristics as primary mouse liver cell but also can be subcultured in vitro. SV40T-immortalized mouse liver cell line can be transfected by pHBV1.3plasmid. After transfected, HBVDNA can replicate in SV40T-immortalized mouse liver cell line, such as that can formate virus replication intermediates including rcDNA, dsDNA and ssDNA and synthesis HBV-specific protein of HBsAg, HBeAg and HBcAg and progeny virus particles that can be secreted out of the cell. So, SV40T immortalized mouse hepatic cell lines can be used as a new suitable for HBV transiently transfected cell model and can provide a new cell model for screening antiviral drug in vitro. It laid the foundation for the establishment of the HBVDNA cross-species transfection liver cells.