OBJECTIVETo study the role of tumor necrosis factor-alpha (TNFalpha) in the anti-replication effects of tetracycline (Tet) on hepatitis B virus (HBV).

METHODSThe Tet-dependent regulatory fragment (TO) was PCR amplified from the pcDNA4TM/TO vector, inserted into the pUC118 cloning vector, and verified by sequencing. The counterpart fragment in the pVITRO3 expression vector, which contains two multiple cloning sites (MCSs), was replaced with the confirmed TO to generate a pVITRO3-TO vector. The Tet repressor (TR) gene from the pcDNA6/TR regulatory vector was incorporated into one MCS of pVITRO3-TO and the TNFalpha gene was subsequently incorporated into the other MCS. The resultant vector, pVITRO3-TOTR-TNFalpha, was transiently transfected into HepG2 cells. TNFalpha expression from the vector was induced by exposure to various concentrations of Tet and measured by enzyme-linked immunosorbent assay to determine the appropriate Tet concentration for experimentation. To investigate whether Tet inhibits TNFalpha expression as a mechanism of its anti-replication activity against HBV, the HepG2.2.15 cell line stably transfected with pVITRO3-TOTR-TNFalpha was used as an HBV replication model. Levels of hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) were detected by immunoassay. HBV DNA level was detected by fluorescence quantitative PCR.

RESULTSThe TNFalpha expression from the newly constructed pVITRO3-TOTR-TNFalpha vector was Tet-controllable in the eukaryotic cells examined. The optimal concentration of Tet for the experimental system was 1.0 mug/ml. HBsAg and HBeAg expression was down-regulated in the HepG2.2.15 cells stably transfected with the pVITRO3-TO-TR-TNFalpha vector. After incubation with Tet for 1, 3 and 5 days, the inhibition rate of HBsAg was 2%, 1.1% and 0, compared to 14.8%, 11.5% and 28.4% in the non-Tet control group. The corresponding inhibition rates of HBeAg were 50.0%, 26.7% and 47.9%, compared to 0.3%, 1.6% and 0.0%, in the control group. HBV DNA levels in the cells and the cell culture supernatants exposed to Tet were decreased by 70.3% and 79.9%, respectively. TNFalpha inhibited production of HBsAg mRNA.

CONCLUSIONA Tet-dependent regulatory fragment double-expressing TNFalpha single vector system was constructed successfully, achieving controllable TNFalpha expression in both transiently transfected eukaryotic cells and stable cell lines. In this HBV cell model system, Tet-induced overexpression of human TNFalpha inhibited HBV DNA replication and reduced HBsAg and HBeAg expression. Inhibition of HBV transcription may be a key role of TNFalpha against HBV replication.

DNA, Viral ; biosynthesis ; Genetic Vectors ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; drug effects ; physiology ; Humans ; Tetracycline ; pharmacology ; Transfection ; Tumor Necrosis Factor-alpha ; genetics ; Virus Replication

OBJECTIVETo construct SD rat immortalized dental follicle cells (rDFC) induced by simian virus 40 large tumor antigen (SV40Tag) gene to provide a reliable cell source for periodontal tissue engineering research.

METHODSThe rDFC was isolated by tissue mass method combined with enzyme digestion method and evaluated by immunohistochemistry. Cell293 were transfected with plasmid pSSR69/pAmpho containing SV40Tag gene by mediating liposome. Normal rDFC were infected with virus-contained supernate and the successfully transfected cell lines were screened with hygromycin, and positive clones were cultured. While non-transfected cells served as negative controls, the cell morphology was observed, the proliferation characteristics was evaluated by calculating cell population. The expression of SV40Tag gene and telomerase in cells was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively. The biological property of immortalized rDFC was assessed with calculating formation rate of flat cloning, soft agar colony formation test and tumor-forming test.

RESULTSMorphology of immortalized rDFC was not different from that of normal rDFC. The RT-PCR results of SV40Tag revealed amplification band at 357 bp, while no band was seen in the normal cells. The expression of telomerase in immortalized rDFC was higher than that in normal rDFC. The two groups had no significant difference in growth curves, but the immortalized rDFC exhibited stronger proliferative activity. No significant differences of formation rate in flat cloning were observed between the immortalized rDFC [34% (33/96)] and normal rDFC at passage four [22% (21/96)] (χ(2) = 3.71, P > 0.05). No cell cloning was seen in soft agar and the tumor formation was not observed in nude mice.

CONCLUSIONSThe rDFC induced by SV40Tag gene could be cultured and passaged in vitro, which retained the stable proliferation and differentiation characteristics and could be used for periodontal tissue engineering research.

Animals ; Antigens, Viral, Tumor ; genetics ; metabolism ; Cell Differentiation ; Cell Proliferation ; Cell Transformation, Viral ; Cells, Cultured ; Dental Sac ; cytology ; immunology ; metabolism ; HEK293 Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Plasmids ; Rats ; Rats, Sprague-Dawley ; Simian virus 40 ; genetics ; immunology ; Telomerase ; metabolism ; Transfection

Carrier Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Gene Library ; Hepatitis B Surface Antigens ; immunology ; metabolism ; Hepatitis B virus ; genetics ; immunology ; Humans ; Immunoprecipitation ; Intracellular Signaling Peptides and Proteins ; metabolism ; Nerve Tissue Proteins ; metabolism ; Viral Proteins ; immunology ; metabolism

OBJECTIVETo study hepatitis B virus (HBV) expression in 3 hepatocytes infected with recombinant adenovirus containing 1.2-copy HBV DNA.a

METHODSA chicken hepatoma cell line and two human hepatocytes were infected by the recombinant adenovirus containing 1.2-copy HBV DNA at 25 pfu/cell. HBV-specific mRNA was detected by RT-PCR 3 days after the infection, and HBsAg and HBeAg were detected by ELISA and HBV DNA by real-time PCR daily after the infection.

RESULTSHBV mRNA expression was detected in all the 3 cells after recombinant adenovirus infection, and the quantities of HBV DNA and HBV antigens in the culture supernatant increased with the passage of time.a

CONCLUSIONInfection with the recombinant adenovirus containing 1.2-copy HBV DNA can mediate HBV infection in the 3 cells in vitro.

Adenoviridae ; genetics ; Animals ; Cell Line, Tumor ; Culture Media, Conditioned ; metabolism ; DNA, Recombinant ; genetics ; DNA, Viral ; genetics ; metabolism ; Gene Expression ; Hepatitis B Antigens ; metabolism ; Hepatitis B virus ; Hepatocytes ; metabolism ; virology ; Humans ; Reverse Transcriptase Polymerase Chain Reaction

BACKGROUND/AIMS: It is essential to develop an in vitro culture model of primary hepatocytes for the study of hepatocellular function and the pathogenesis of hepatitis C virus (HCV) infection. In this study, we have established the immortalized primary human hepatocyte (IPHH) and performed in vitro culture of HCV derived from human patient. METHODS: Primary human hepatocytes were isolated from surgically resected liver tissue and then were immortalized by transfection with the SV40 large T antigen. The characterization of the IPHH during culture was analyzed by immunocytochemistry, RT-PCR, Western blot, ELISA, and soft agar assay. Next, sera and/or liver tissue homogenates from surgically resected liver tissues of patients with HCV infection were inoculated for the culture of HCV in IPHH. After HCV RNA extraction from IPHH and culture media, positive or negative stranded HCV RNA was examined by specific nest RT-PCR. RESULTS: IPHH expressed liver-associated proteins but did not express alpha-fetoprotein. Also IPHH showed ammonia removal activity. With regard to its malignant potential, colony formation in soft agar assay was not observed. Next, positive and negative stranded HCV RNAs in IPHH infected with patient's sera plus liver tissue homogenates were clearly detected whereas those in IPHH infected with only patient's sera were not detected. CONCLUSIONS: These results demonstrated the phenotypic characteristics of IPHH and the feasibility in vitro culture system of HCV infected human samples. This system might be useful for study of pathogenesis of HCV infection or hepatocyte-based applications.
Antigens, Viral, Tumor/genetics ; Base Sequence ; Carcinogenicity Tests ; Cell Culture Techniques ; Cells, Cultured ; Cells, Immobilized ; Hepacivirus/isolation & purification/*physiology ; Hepatocytes/metabolism/physiology/*virology ; Humans ; Liver Function Tests ; Models, Biological ; RNA Probes ; RNA, Viral/analysis ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate whether there were particular HBx gene mutations associated with hepatocellular carcinoma (HCC) development in patients.

METHODSThe HBx genes were examined in 51 paraffin-embedded tumor tissue samples from patients with HCC and 25 serum samples from HBV carriers from southern China by nested polymerase chain reaction (PCR), single-stranded conformational polymorphism analysis, heteroduplex analysis and DNA sequencing. The HBx genes with deletion variations (HBx-d382, HBx-d431) from tumor tissues were cloned and transfected into QSG7701 cells. Then, the biological characteristics of the transfected cells were analyzed in nude mice by MTT assay, soft agar colony formation assay, flow cytometry and xenografting.

RESULTSDeletion mutation and point mutation were found in the HBx genes of HCC tumor tissues, and there were some differences between the HBx gene mutations in genotype B and those in genotype C. More mutations were found in genotype C than those in genotype B (t=-2.522, P < 0.05), but the deletion variations (HBx-d382, HBx-d431) were detected in genotype B HBV from HCC liver tissues. The HBx genes with deletion variations (HBx-d382, HBx-d431) were recombinant with pcDNA3 and transfected into QSG7701 cell lines successfully, which established four permanent transfected QSG7701 cell lines, including pcDNA3/HBx-d382/QSG7701, pcDNA3/HBx-d431/QSG7701, pcDNA3/HBx/QSG7701, and pcDNA3/QSG7701. pcDNA3/HBx-d382/QSG7701 and pcDNA3/HBx-d431/QSG7701 grew faster and had more potential colony formative activity than those of pcDNA3/QSG7701. Moreover, pcDNA3/HBx-d382/QSG7701 and pcDNA3/HBx-431/QSG7701 cells inoculated in nude mice produced tumors more rapidly than those of pcDNA3/HBx/QSG7701, and pcDNA3/QSG7701. The volumes of the tumors in nude mice were also obviously larger in pcDNA3/HBx-d382 and pcDNA3/HBx-d431 groups than those in pcDNA3/HBx/QSG7701 and pcDNA3/QSG7701 groups.

CONCLUSIONOur results suggest that HBx gene mutations occur frequently in HCC tissues, and the deletion at nt382-400 of the HBx gene might play a role in carcinogenesis of HCC in southern China.

Adult ; Aged ; Animals ; Base Sequence ; Carcinoma, Hepatocellular ; virology ; Cell Line, Tumor ; DNA, Viral ; genetics ; Female ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; genetics ; Humans ; Liver Neoplasms ; virology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Middle Aged ; Point Mutation ; Polymorphism, Single-Stranded Conformational ; Sequence Analysis, DNA ; Sequence Deletion ; Trans-Activators ; genetics ; Transfection

OBJECTIVETo search for and verify some common B cell epitopes in the core proteins of woodchuck hepatitis virus and human hepatitis B virus.

METHODSMonoclonal antibodies against both core proteins of woodchuck hepatitis virus (WHV) and human hepatitis B virus (HBV) were prepared by inoculating Balb/c mice with denatured recombination WHV and HBV core proteins. ELISA and immunoblotting assays for WHcAg and HBcAg were carried out by using these antibodies. Immunohistochemistry was carried out with liver tissue sections of both WHV-infected woodchucks and chronic HBV-infected patients. The epitopes were mapped with the mouse mAbs (6D1 and 1H4) by using a panel of 24 16mer overlapping peptides covering the entire WHcAg. The amino acid sequences of WHcAg and HBcAg were compared.

RESULTSCross-reactions were observed between mAbs (6D1 and 1H4) and WHcAg and between Mabs and HBcAg/HBcAg in ELISA and immunoblotting assay. Liver tissue sections of both WHV-infected woodchucks and chronic HBV-infected patients could be stained specifically by mAbs. The epitopes were mapped at aa1-8 (6D1) and aa125-140 (1H4) of the core proteins of both WHV and HBV by using ELISA assay. WHcAg and HBcAg share similar amino acids sequences at aa1-8 and aa125-140 respectively.

CONCLUSIONThe core proteins of woodchuck hepatitis virus and human hepatitis B virus share common linear B cell epitopes which span aa1-8 and aa125-140 respectively.

Animals ; Antibodies, Monoclonal ; biosynthesis ; B-Lymphocytes ; immunology ; Cell Line, Tumor ; Cross Reactions ; Epitopes, B-Lymphocyte ; immunology ; Hepatitis B Core Antigens ; immunology ; Hepatitis B Virus, Woodchuck ; genetics ; immunology ; Hepatitis B virus ; genetics ; immunology ; Humans ; Marmota ; Mice ; Viral Core Proteins ; immunology

OBJECTIVEo study the replication of hepatitis B virus (HBV) in HepG2 cells infected with Ad-1.2 HBV.

METHODSHepG2 cells were transfected with adenovirus containing 1.2 copies of HBV DNA. The expression of HBV antigens were detected in the culture medium by means of enzyme-linked immunosorbent assay (ELISA), and the covalently closed circular DNA (cccDNA) in the cells was extracted with plasmid extraction kit and detected by real-time PCR with selective primer after treatment with mung bean nuclease.

RESULTSHBsAg, HBeAg and HBV cccDNA were all detected in HepG2 cells after tranfection with Ad-1.2 HBV. HBV cccDNA was detected 1 day after the infection, reaching the peak level 4 days after infection.

CONCLUSIONAd-1.2 HBV-infected cells can serve as the model for screening and evaluation of antiviral agents.

Adenoviridae ; genetics ; Calibration ; Cell Line, Tumor ; DNA, Complementary ; genetics ; metabolism ; DNA, Viral ; genetics ; metabolism ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; immunology ; metabolism ; physiology ; Humans ; Polymerase Chain Reaction ; Time Factors ; Transfection ; Virus Replication

OBJECTIVETo screen efficient siRNA for inhibiting hepatitis B virus using the technique of PCR-based tRNA(val) Pol III-shRNA expression cassettes (SECs).

METHODSBased on core gene sequence of HBV, five target sites of siRNA were designed. tRNAval Pol III-shRNA expression cassettes produced by one-step overlapping extension PCR strategy were co-transfected with HBV C gene and pC-EGFP plasmid into AD293 cells respectively. Forty-eight hours after transfection, fluorescence of HBVC-GFP protein was detected by fluorescence-activated cell sorting (FACS); HBV C mRNA was detected by semi-quantitative RT-PCR. HBV-producing HepG2. 2. 15 cells were transfected with selected SECs for 72 h, HBsAg and HBeAg in the cell culture medium were detected by radioimmunoassay assay (RIA). HBV pgRNA from cell total RNA was detected by semi-quantitative PCR.

RESULTCo-transfection with pC-GFP plasmid and SECs into AD293 cells resulted in inhibition expression of HBV C gene and decrease of EGFP fluorescence intensity. SEC-492i showed most significant inhibition effect on HBV C-EGFP expression compared with other SECs. Selected SEC-492i or SEC-282i targeting core gene could efficiently decrease expression of HBeAg and the level of HBV pgRNA in a dose-dependent manner. SEC-492i inhibited HBV replication and antigen expression in a more efficient way than SEC-282i at the same final concentration.

CONCLUSIONThe expressed shRNA, which targets sites on HBV C mRNA in 492i, is to have having most efficient RNAi effect. tRNAval Pol III-shRNA expression cassettes produced by one-step overlapping extension PCR strategy should be useful for identification of optimal siRNA.

Base Sequence ; Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; Cells, Cultured ; Embryo, Mammalian ; Green Fluorescent Proteins ; genetics ; Hepatitis B Core Antigens ; biosynthesis ; genetics ; Hepatitis B e Antigens ; biosynthesis ; genetics ; Hepatitis B virus ; genetics ; Humans ; Kidney ; cytology ; Liver Neoplasms ; pathology ; Molecular Sequence Data ; Polymerase Chain Reaction ; RNA, Messenger ; genetics ; RNA, Small Interfering ; RNA, Transfer, Val ; genetics ; RNA, Viral ; genetics ; Transfection

OBJECTIVETo investigate whether simian virus 40 (SV40) was related to patients of malignant mesothelioma in China.

METHODSParaffin-embeded samples of 17 patients with malignant mesothelioma were collected. After isolation of DNA from paraffin blocks, polymerase chain reaction (PCR) analyses were performed using three different sets of primer for detection of SV40 large T antigen gene. These samples were also immunohistochemically evaluated for expression of SV40 TAg protein with two different anti-SV40 Tag (Pab101 and Ab-2).

RESULTSOnly one of the three primer pairs successfully amplified SV40 genome in three malignant mesothelioma samples. No immunopositive staining for SV40 TAg was found in any of the samples.

CONCLUSIONSThe study shows that malignant mesothelioma in China may be independent of SV40 infection.

Adult ; Aged ; Antigens, Viral, Tumor ; genetics ; metabolism ; China ; Female ; Host-Pathogen Interactions ; Humans ; Immunohistochemistry ; Male ; Mesothelioma ; pathology ; virology ; Middle Aged ; Polymerase Chain Reaction ; Polyomavirus Infections ; pathology ; virology ; Simian virus 40 ; genetics ; immunology ; physiology ; Tumor Virus Infections ; pathology ; virology ; Young Adult