OBJECTIVEPersistent replication of hepatitis B virus (HBV) is one of the major obstacles in HBV infection treatment. Reduction or clearance of HBV propagation would be one of the aims of HBV therapy. The drugs approved in clinical used such as nucleotide analogs or interferon, were limited effects on HBV replication. The newly developing gene therapy method, dominant negative mutants, were be used as new promising HBV therapy strategy, and a dominant negative mutant of HBVX gene pRev X-GFP which we have reported in our previous study has some effects both on HBV replication and expression in transient expression, but the effects were interfered by persistent secretion of HBV in HepG2 2.2.15 cells without transfection pRev X-GFP in the experiment. To make sure the effects of dominant negative mutant of pRev X-GFP, we established a HBX DN stable express cell clone, and evaluated the effects of HBX dominant negative mutant on HBV replication.

METHODSThe X gene mutant, in which a specific point mutation of 3'-end ATG to AAG and fused with human green fluorescence protein (GFP) were cloned into pRev TRE vector, assigned to pRev HBX-GFP dominant mutant (pRev X-GFP). And the plasmid contains the wild type X gene or GFP gene was cloned into the same vector to construct pRev Xwt, pRev GFP constructs. All the constructs then transfected into HepG2 2.2.15 cells by liposome. After 7 days resistance selection of hygromycin (300 microg/ml), and cell clones which stable expression HBX-GFP, HBXwt, GFP were obtained. After reseeding of 106 cells of each clones in 12 wells with a 12 well cell plate and another 12 wells 2.2.15 cell were serve as blank control. The cells and media were harvested after cultured in DMEM with 10% FBS for 3 days. HBV-related DNA was assayed by dot blot and Southern blot.

RESULTSThe 100% expression of pRev HBX-GFP, GFP and wild type X constructs were obtained. The stable expressed HBX-GFP can significantly reduce HBV DNA level both in cell media and cells by dot blot and Southern blot analysis, but not for pRev Xwt and pRev GFP.

CONCLUSIONThe dominant negative mutant pRev HBX-GFP can significantly inhibit the HBV gene expression. It also suggested that X gene might be one of promising target for HBV gene therapy.

Carcinoma, Hepatocellular ; pathology ; Cloning, Molecular ; DNA Replication ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Hepatitis B virus ; genetics ; physiology ; Humans ; Liver Neoplasms ; pathology ; Point Mutation ; Trans-Activators ; genetics ; Transfection ; Tumor Cells, Cultured ; Virus Replication

Cell Line ; DNA, Viral ; genetics ; Genome, Viral ; genetics ; Hepatitis B ; pathology ; virology ; Hepatitis B virus ; genetics ; Humans ; Liver Neoplasms ; pathology ; virology ; Transfection

Genetic Engineering ; Genetic Therapy ; Green Fluorescent Proteins ; Hepatitis B virus ; genetics ; physiology ; Luminescent Proteins ; genetics ; Mutation ; Trans-Activators ; genetics ; Virus Replication

OBJECTIVEIn order to explore the roles of Huoxueruanjian compound on liver fibrogenesis and its molecular mechanism, this paper has investigated the Influence of blood serum with such traditional Chinese medicine compound on the expression of Smad3, Smad7 and procollagen alpha2(I) gene in hepatic stellate cell (HSC).

METHODSHSC-T6 was deal with different Concentration of blood serum medicine with Heluoshugan which was made by routine way. Then expression change of Smad3, Smad7 and procollagen alpha2(I) mRNA among each groups were observed by RT-PCR. Furthermore, the expression change of Smad3 protein were examined by Western blot.

RESULTSExpression of Smad3 and procollagen alpha2(I) mRNA as well as Smad3 protein had been downregulated after treating with blood serum medicine of Heluoshugan (P<0.01, P<0.05, respectively). The expression of procollagen alpha2(I) mRNA changed at the same tendency as those of Smad3. The role of blood serum medicine was significant difference between different concentration, P<0.05. And the expression of procollagen alpha2(I) mRNA changed in concentration-dependent manner. Blood serum medicine has no effects on the Smad7 mRNA.

CONCLUSIONThe anti-fibrosis roles of HuoXueruanjian Compound maybe influence the function of TGF-beta and Smad by nonspecific action, thereby inhibit the transcription of procollegan alpha2(I) mRNA and decrease the production of ECM. As regards Smad3, it may be facilitating the development of liver fibrosis when its expression increases. Otherwise, it manifest with anti-fibrosis role.

Animals ; Collagen Type I ; genetics ; DNA-Binding Proteins ; genetics ; Liver Cirrhosis ; drug therapy ; etiology ; pathology ; Medicine, Chinese Traditional ; RNA, Messenger ; analysis ; Rats ; Smad3 Protein ; Smad7 Protein ; Trans-Activators ; genetics

OBJECTIVETo evaluate the mutations of lamivudine-resistance using oligonucleotide microarray in hepatitis B virus (HBV) infected patients.

METHODSA randomized clinical trial was conducted on 20 lamivudine-treated patients for 18 months and 10 patients as controls. The serum HBV DNA was amplified by PCR and the lamivudine-resistance mutations in YMDD region were assayed by 4 sites microarray developed before.

RESULTSThis microarray could clearly differentiate the wide-type from mutated-type HBV with lamivudine-resistance mutations. The rate of mutations in YMDD region increased with the time of lamivudine treatment (chi2=6.69, P<0.01). The most common mutated type was M539V+L515M and next M539I. Continuous administration of lamivudine was no benefit for inhibiting the replication of HBV with YMDD mutation but helpful for wide-type HBV.

CONCLUSIONThe routine serum HBV DNA assay by PCR may introduce prejudice in monitoring HBV inhibitory effect by lamivudine, while the microarray technique can avoid this and is one of the best ways to monitor the lamivudine-resistance mutations in HBV. There is no effect of lamivudine on HBV with YMDD mutation in clinical practice.

Adult ; Antiviral Agents ; pharmacology ; therapeutic use ; Drug Resistance, Viral ; genetics ; Female ; Hepatitis B virus ; drug effects ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Lamivudine ; pharmacology ; therapeutic use ; Male ; Middle Aged ; Oligonucleotide Array Sequence Analysis ; Point Mutation ; genetics ; Polymerase Chain Reaction

Forkhead Transcription Factors ; genetics ; Gene Expression Regulation ; Humans ; Procollagen ; genetics ; Transcription, Genetic

Antiviral Agents ; pharmacology ; Dideoxyadenosine ; analogs & derivatives ; pharmacology ; Hepatitis B virus ; drug effects ; Virus Replication ; drug effects

OBJECTIVETo establish a highly expressing and replicating hepatitis B virus (HBV) genome transgenic mouse models for screening anti-HBV drugs and investigating the pathogenesis of hepatitis B.

METHODSElongated HBV genome as the investigated gene was transducted into the pronuclei of the fertilized eggs of mice by the technique of microinjection, then the eggs were transplanted into the oviducts of the pseudopregnant mice. All the newborn mice were screened and identified by PCR and Southern blot detecting genomic DNA in tail tissue, then the positive mice were examined plasma HBsAg, HBeAg by ELISA and plasma HBV DNA by Southern blot.

RESULTSAmong the 61 offsprings, 18 were positive for tail tissue HBV DNA examination, 7 of which were positive for replication and expression detection.

CONCLUSIONTransgenic mice with elongated HBV genome possess high efficiency of replication and expression, which can be used for further investigation.

Animals ; DNA Replication ; DNA, Viral ; genetics ; Disease Models, Animal ; Genome, Viral ; Hepatitis B ; virology ; Hepatitis B Surface Antigens ; genetics ; Hepatitis B e Antigens ; genetics ; Hepatitis B virus ; genetics ; physiology ; Mice ; Mice, Transgenic ; genetics ; Virus Replication

Gene Expression ; Hep G2 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Liver Neoplasms ; genetics ; metabolism

OBJECTIVETo explore the effect and the molecular targets of anti-hepatitis B virus (HBV) by beta-L-D4A in vitro.

METHODS2.2.15 cells were cultured and treated with various concentrations of beta-L-D4A for 6 hours, then the effect of anti-HBV was examined by Southern blot and the replicating core particles from the cells were isolated. The endogenous polymerase reaction and activity gel experiment were performed to monitor the activities of the DNA polymerase and reverse transcriptase.

RESULTSThe replication of HBV DNA was inhibited in a dose-dependent manner. The endogenous polymerase reaction showed both the two enzymatic activities were irreversibly inactivated in a concentration -dependent manner, with IC50 at 0.51 micromol/L and 0.55 micromol/L, respectively. But the activities of DNA polymerase and reverse transcriptase were found to remain active by activity gel with exogenous templates.

CONCLUSIONSThe mechanism of inhibiting HBV replication by beta-L-D4A may be in that either the DNA replication priming is blocked or the elongation of DNA chain is terminated irreversibly.

Antiviral Agents ; pharmacology ; DNA, Viral ; biosynthesis ; Dideoxyadenosine ; analogs & derivatives ; chemistry ; pharmacology ; Dose-Response Relationship, Drug ; Hepatitis B virus ; drug effects ; enzymology ; physiology ; Humans ; Liver Neoplasms ; pathology ; Nucleic Acid Synthesis Inhibitors ; Tumor Cells, Cultured ; Virus Replication ; drug effects