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Objective: To investigate the biological role of auto-induced expression of hepatitis C virus (HCV) core protein (protein C) using a recombinant protein in an in vitro cell-based system., Methods: The PCR-amplified full-length HCV protein C gene (573 bp) was inserted into the pET28a prokaryotic expression vector. The recombinant plasmid was transformed into BL21(DE3)pLysS E. coli to achieve high-concentration expression of the recombinant C protein by auto-induction. The recombinant protein C was purified by Ni-NTA affinity chromatography, and tested in a protein binding assay for its ability to bind the HCV NS3 protein., Results: The transformed E. coli produced a large amount of recombinant protein C, as detected in the sonicated supernatant of the bacteria culture. The antigenic reactivity of the recombinant protein C was confirmed by western blotting. However, the recombinant protein C could not be purified by Ni-NTA affinity chromatography, but co-precipitated with the HCV NS3 protein., Conclusion: Soluble recombinant protein C was successfully expressed by auto-induction, and shown to interact with the HCV NS3 protein, which provides a novel insight into the putative biological activity of this factor in HCV-related molecular processes. Future studies of this recombinant HCV protein C's crystal structure and antigenicity may provide further clues to its biological function(s) and potential for clinical applications.

Objective: To establish a detection method for HBV drug-resistant mutations related to lamivudine, adefovir and entecavir by optimization and assessment of reverse hybridization system., Method: 26 degenerated probes covering 10 drug-resistant hotspots of 3 drugs were synthesized and immobilized on the same positively charged nylon membrane. PCR products labeled with digoxigenin were hybridized with corresponding probes. To improve the sensitivity and specificity, 4 reaction steps of reverse hybridization were optimized including the number of labeled digoxigenin, the energy intensity of UV cross-linking, hybridization and stringency wash conditions. To prove the feasibility, the specificity, sensitivity and accuracy of this system were assessed respectively., Result: Sensitive and specific results are obtained by the optimization of the following 4 reaction steps: the primers labeled with 3 digoxigenin, energy intensity of UV cross-linking for 1500 x 0.1 mJ/cm², hybridization at 42 degrees C and stringency wash with 0.5 x SSC and 0.1% SDS solution at 44 degrees C for 30 min. In the assessment of system, the majority of probes have high specificity. The quantity of PCR product with a concentration of 10 ng/μl or above can be detected by this method. The concordant rate between reverse hybridization and direct sequencing is 93.9% in the clinical sample test., Conclusion: Though the specificity of several probes needs to be improved further, it is a simple, rapid and sensitive method which can detect HBV resistant mutations related to lamivudine, adefovir and entecavir simultaneously. Due to the short distance between 180 and 181, likewise 202 and 204, the sequence of the same probe covers two codon positions, and hybridization will be interfered by each other. To avoid such interference, the possible solution is that probes are designed by arranging and combining various forms of two near codons.

Objective: To investigate the evolution of hepatitis B virus (HBV) quasispecies in one patient during lamivudine (LAM) monotherapy and switching to entecavir (ETV) rescue treatment., Methods: Serum samples were taken at seven different time points during antiviral therapy (0, 24, 48, 60, 72, 96, 152 weeks, respectively), the HBV DNA polymerase gene was amplified, cloned and sequenced to analyze the amino acid substitutions within HBV DNA polymerase gene and distribution of virus quasispecies. Quantitative detection of the HBV wild strains and total virus was performed by amplification refractory mutation system real-time PCR (ARMS-PCR)., Results: Three mutation patterns detected during antiviral therapy in the patient: rtM204V, rtM204V+rtL180M and rtM204I. The HBV quasispecies were found always in dynamic variation. The HBV populations were completely replaced with the LAM-resistant variants when the viral breakthrough was encountered during LAM monotherapy. Interestingly, the wild-type variants presented gradually dominant (79.3%) with the decline of HBV DNA load after switching to ETV rescue administration. ARMS-PCR results showed that the wild-type variants account ed for 68.55% of the HBV populations at baseline and this proportion declined to 0.21% when the viral breakthrough emerged under LAM therapy. The wild-type variants gradually increased from week 24 after switching to ETV rescue therapy and the proportion of HBV wild-type variants in the population fluctuated between 16.01% to 26.93%., Conclusions: The distribution of virus quasispecies were always in dynamic variation during sequential therapy with nucleotide analogs in chronic hepatitis B patients. Different patterns of dynamic HBV quasispecies may have different contribution in ETV resistance in LMV refractory patients with ETV administration.

Objective: To investigate the effect of HS3ST3B1 on hepatitis B virus (HBV) replication., Methods: HepG2 cells were classified into 7 groups according to the plasmids transfected: (1) Blank group, no plasmid transfected; 2. Positive control, transfected with pCH9-HBV which permits HBV replication; (3) Negative control, transfected with pCH9-HBV + pcDNA3.1 + pTZU6+1; (4) Treatment A, transfected with pCH9-HBV + pCDNA3.1-HS3ST3B1 + pTZU6+1; (5) Interference A, transfected with pCH9-HBV + pCDNA3.1-HS3ST3B1 + psh1126 (a plasmid to interfere HS3ST3B1 expression); (6) Treatment B, transfected with pCH9-HBV + pTZU6+1; (7) Interference B, transfected with pCH9-HBV + psh1126. The levels of HBV DNA were detected in the above groups by Southern blotting. HBV total RNA of Negative control, Treatment A and Interference A were quantified by Real-time PCR to determine the influence of HS3ST3B1 over-expression on the HBV RNA transcription. The activity of the four HBV promoters [core promoter (cp), x promoter(xp), surface antigen promoter1(sp1), surface antigen promoter2 (sp2)] were assayed by Dual-Luciferase Reporter Assay System. The data was analyzed using one way ANOVA, with P < 0.05 indicating statistically meaningful difference., Result: Southern blot data revealed the level of HBV DNA in Treatment A and Interference A accounted for 10% +/- 2% and 31% +/- 4% of that in control. Compared with control, a statistical difference existed between Treatment A and Control, with F value equalling to 20.8 and P value equalling to 0.034 respectively. A statistical difference also existed between Interfere A and Treatment A, with F value equalling to 24.9 and P value equalling to 0.021 respectively. The level of HBV DNA in Experiment B was raised by 130% +/- 11% as compared to that in Interference B, and the levels of HBV DNA showed a dose-dependent decrease when H7 cells were transfected with 0.5, 1.0, 1.5 microg pCDNA3.1-HS3ST3B1 respectively. Statistical differences existed between control and H7 transfected with different dose of pCDNA3.1-HS3ST3B1, with F values equalling to 22.7, 20.3, 26.5 and P values equalling to 0.029, 0.041 and 0.015 respectively. Real-time PCR revealed that the HBV total RNA in Treatment A accounted for 17.0% +/- 2.7% of that in control and there was a statistical difference between Treatment A and control, with F value equalling to 25.6 and P value equalling to 0.018. In addition, HBV DNA in Interference A was restored to 74.0% +/- 3.9% of that in control, and there was also a statistical difference between Treatment A and Interference A, with F value equalling to 21.3 and P value equalling to 0.032. However, the down regulation of HBV total RNA had nothing to do with HBV promoters activity., Conclusion: HS3ST3B1 can inhibit HBV replication and reduce the level of HBV total RNA, but the downregulation of HBV total RNA may not be the result of direct interaction of HS3ST3B1 and HBV promoters.

Objectives: To establish a method for simultaneous detection of HBV resistant mutations associated with three kinds of nucleoside analogues., Methods: According to 981 HBV complete sequences in GenBank, two pairs of conserved primers labeled with digoxigenin were synthesized to amplify the region of HBV reverse transcriptase. To detect non-synonymous amino acid substitutions associated with lamivudine, adefovir and entecavir, 26 specific oligonucleotide probes covering ten different codon positions, I169T, V173L/G, L180M, A181T/V, T184G, S202I/G, M204V/I, Q215S, N236T and M250V/I/L were synthesized and immobilized on nylon membranes charged positively. The oligonucleotide probes immobilized on nylon membranes were then hybridized with PCR products labeled with digoxigenin to detect three drug-resistant mutations. In order to observe specificity and accuracy of probes, HBV wild-type, resistant reference strains and patients serums were assayed by reverse hybridization technique, respectively., Results: The specific probes of 10 codon positions related to HBV wild-type and resistant reference strains, including I169T, V173L, L180M, A181T, T184G, S202I, M204V, Q215S, N236T, M250V, were distinguished effectively by reverse hybridization method. The results results of 37 samples applicated the method were in accordance with that Of DNA sequencing., Conclusion: Reverse hybridization technique can be applied to detect HBV resistant mutations associated with Lamivudine, Adefovir and Entecavir rapidly and accurately.

Objective: To investigate if inflammatory stress enhances liver lipid accumulation via SREBPs mediated dysregulation of low density protein receptor (LDLr) expression in apolipoprotein E, scavenger receptors class A and CD36 triple knockout (ApoE/SRA/CD36 KO) mice., Methods: 16 Male ApoE/SRA/CD36 KO mice were subcutaneously injected with 0.5 ml 10% casein or PBS. The mice were fed a Western diet (Harlan, TD88137) containing 21% fat and 0.15% of cholesterol for 14 weeks. Animals were sacrificed and blood samples were collected. The serum amyloid A (SAA), IL-6, total cholesterol (TC), LDL and high density protein (HDL) were assayed. The lipid accumulation in liver was evaluated by Oil Red O staining. The mRNA and protein expression of SREBP-2, SREBPs cleavage activating protein (SCAP) and LDLr were analyzed by Real-Time Polymerase Chain Reaction (RT-PCR) and immunohistochemistry staining., Results: Blood levels of SAA [(26.60+/-3.24) ng/ml vs (14.35+/-1.73) ng/ml, P < 0.01] and IL-6 [(36.37+/-2.20) pg/ml vs (18.02+/-4.87) pg/ml, P < 0.01] were higher, while TC [(7.72+/-1.70) mmol/L vs (13.23+/-3.61)mmol/L, P less than 0.01], LDL-cholesterol [(2.94+/-0.44) mmol/L vs (9.28+/-3.66) mmol/L, P less than 0.01] and HDL cholesterol [(2.24+/-0.63) mmol/L vs (4.13+/-0.42) mmol/L, P less than 0.01] were lower in inflamed mice compared to controls. ORO staining showed that lipid accumulation in the liver was more extensive in inflamed group despite lower blood lipid levels. Meanwhile, Real Time PCR data showed inflammation induced the expression of LDLr (4.56 fold), SCAP (3.14 fold) and SREBP-2 (14.72 fold) in liver. Immunohistochemical staining also indicated increased proteins expression in the liver, which was consistent with mRNA data., Conclusions: Inflammation causes lipid accumulation in liver via disrupting SREBP-2 and LDLr expression.

Objective: To compare the sensitivity of different hepatocellular carcinoma (HCC) cell lines (HepG2, QGY7701, HepG2.2.15) and the normal liver cell line L02 to 5-aza-dC, an DNA methyltransferase inhibitor, and to explore the relationship between global DNA methylation level and the sensitivity to 5-aza-dC., Methods: HepG2, QGY7701, HepG2.2.15 and L02 cells were treated with 5-aza-dC at different concentration, cell proliferation was measured by MTT method, cell apoptosis was detected by measuring caspase 3 activity and cellular DNA fragmentation ELISA., Results: Compared to HepG2 and QGY7701 cells, HepG2.2.15 were less sensitive to the treatment of 5-aza-dC; the normal liver cell line L02 was less sensitive to 5-aza-dC than the HCC cell lines., Conclusions: The sensitivity to 5-aza-dC of HCC cell lines and normal liver cells is not correlated with the global DNA methylation level.

Objective: To investigate the effect of adiponectin on hepatocyte steatosis., Methods: L02 cells were transfected with pEGFP-N1-AdipoQ, a plasmid encoding pEGFP-adiponectin fusion protein, or pEGFP-N1. Lipid droplets in the hepatocytes were observed by oil red staining at 72 h. The contents of TG, FFA and glycerol in hepatocytes were measured., Results: Compared to cells transfected with pEGFP-N1-AdipoQ plasmid, much more lipid droplets were observed in cells transfected with pEGFP-N1 plasmid. TG, FFA and glycerol contents in L02 cells and L02/pEGFP-N1 cells were significantly higher than those in L02/pEGFP-N1-AdipoQ cells., Conclusions: Overexpression of adiponectin prevent hepatocyte steatosis.

Objective: To explore the mechanism for adefovir dipivoxil (ADV) resistance occurred in chronic hepatitis B patients of a series of phase III clinical trails., Methods: 30 resistant HBV strains were selected out from 177 cases of ADV treated chronic hepatitis B patients. HBV polymerase RT region were amplified by nested PCR and analyzed with the standard nucleotide sequence of HBV strains deposited in GeneBank., Results: 21 out of 30 HBV strains were primary resistant strains, among them 5 HBV strains (23.8%, 5/21) had the polymorphism site of rtN118H. While the other 9 HBV strains showed secondary resistance, variations in conservative region C (rtM207V) and other non-conservative regions were found. The classic mutation sites such as rtN236T and rtA181V/T were not found., Conclusions: Polymorphism site of rtN118H might be responsible for HBV primary resistance to ADV therapy. rtM207V variation in HBV RT C domain and other variation sites might play a role in HBV secondary resistance to ADV treatment, and natural resistant quasispecies may be the basis for the ADV quick resistance. These conclusions await further confirmation by phenotype test.

Objective: To investigate the effect of Hyper-IL-6 on the proliferation of L-02 cells., Methods: The recombinant lentiviral vectors encoding Hyper-IL-6 (FIV-Hyper-IL-6), IL-6 (FIV-IL-6) and the lentiviral (FIV) were prepared using the FIV-based lentiviral vectors and the packaging system. The titer of FIV-Hyper-IL-6, FIV-IL-6 and FIV was tested with puromycin. L-02 cells were infected with FIV-Hyper-IL-6, FIV-IL-6, FIV, or mock-infected. The growth rate of L02 cells was analyzed with MTT at different time points after infection. The changes of Haptoglobin in L-02 cells were analyzed with RT-PCR., Results: FIV-Hyper-IL-6, FIV-IL-6 and FIV were successfully constructed, and the titer was 107 pfu/ml. 48 hours after infection, the absorbance of the cells infected with FIV-Hyper-IL-6 was 0.6267+/-0.0256, and the absorbances of FIV-IL-6 infected cells, FIV infected cells and mock-infected cells were 0.5563+/-0.0112, 0.5040+/-0.0078 and 0.4790+/-0.0201, respectively. There were significant differences between the FIV-Hyper-IL-6 group and the other groups (F = 41.09, P less than 0.01). The ratio of the absorbance between haptoglobin mRNA and beta-actin was 0.7030+/-0.0106, 0.3355+/-0.0093, 0.1145+/-0.0076 and 0.1143+/-0.0153, respectively, in FIV-Hyper-IL-6 infected cells, FIV-IL-6 infected cells, FIV infected cells and mock-infected cells. There were significant differences between the FIV-Hyper-IL-6 group and the others (q = 57.5007, P less than 0.01)., Conclusion: Compared with IL-6, Hyper-IL-6 is more potent to stimulate proliferation and induce the expression of Haptoglobin in L-02 cells.

Objective: To investigate the role of HBV genotypes on their response to adefovir dipivoxil (ADV) antiviral therapy., Methods: HBV genotypes from 177 HBeAg-positive chronic hepatitis B (CHB) patients were identified and the patients were treated with ADV 10 mg per day for 48 weeks. The clinical data in terms of serum HBV DNA seroclearance, mean HBV DNA reduction (log value), HBeAg loss, anti-HBe seroconversion and serum ALT of those patients were analyzed against their HBV genotypes., Results: Genotype B and genotype C were found in 102 and 65 cases, respectively. The mean HBV DNA reduction in patients with genotype B and genotype C at their treatment times of 12, 24 and 48 weeks was 2.2 log10copies/ml, 2.1 log10copies/ml (P more than 0.05), 2.7 log10copies/ml, 2.4 log10copies/ml (P more than 0.05) and 3.6 log10copies/ml, 3.1 log10copies/ml (P less than 0.05), respectively. At the end of the therapy (48 weeks), 43 (42.2%) patients with genotype B HBV infection and 22 (33.8%) patients with genotype C HBV infection had achieved HBV DNA seroclearance (P less than 0.05)., Conclusions: Our results suggest that genotype B HBV has a better virological response to ADV therapy in HBeAg-positive chronic hepatitis B patients than that of genotype C. Longer terms of ADV treatment are needed to confirm this conclusion.

Objective: To establish a set of suitable and reliable methods for HBV genotyping and to study the distribution of HBV genotypes., Methods: Type-specific nucleotides were searched through alignment of S genes (more than 1000 sequences) listed in GenBank. Then, type-specific primers were designed and type-specific primer PCR was used to genotype the 238 HBV strains. S genes of the untyped strains were further amplified and sequenced to find out their genotypes with type-specific nucleotide analysis., Results: All the 238 HBV strains were genotyped. 159 (66.8%) cases were genotype B, 69 (28.9%) were genotype C, 6 (2.5%) were mixtures of genotypes B and C and 4 (1.6%) were mixtures of genotypes B and D. No genotypes of A, E, F, G, and H were found., Conclusion: Genotypes B and C are the most common types for HBV strains. Mixtures of genotypes B and C or genotypes B and D coinfection rarely existed. There is no relationship between the gender of the patients and HBV genotypes (X2 = 0.794, P more than 0.05).

Objective: To learn the effect of hepatitis B virus (HBV) sequence nt250-453 on the HBV X promoter., Methods: A plasmid pNRE-XP which contains the NRE and the HBV X promoter was constructed to co-transfect HepG2 cell line with plasmid RL-TK. The firefly luciferase activity and mRNA expression of the firefly luciferase gene were both detected. Then, nt250-453 of HBV was removed from LJ196, which contained HBV full genes. The mutated plasmid LJ196 and plasmid LJ96 which provided core protein and the viral DNA polymerase were used to co-transfect HepG2 cell line. Reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the X gene mRNA level., Results: The activity of firefly luciferase and the expression of firefly luciferase gene mRNA were both down-regulated in the presence of the NRE, while the HBV X gene mRNA expression increased as it was removed from the HBV genes., Conclusion: This study demonstrates that nt250-453 of HBV acts as a novel negative regulatory element which could suppress the HBV X promoter activity.

Objective: To study the effect of HCV NS5A protein on HCV IRES-dependent translation in HepG2 cells., Methods: HepG2 cells were co-transfected with a plasmid vector containing a bicistronic transcript carrying Renilla luciferase and firefly luciferase genes separated by HCV IRES sequences, and an expressing vector producing the NS5A protein. The luciferase activity and the mRNA of the luciferase gene were then detected. The NS5A expression was confirmed by fluorescence microscopy., Results: HCV NS5A protein was detected in the cytoplasm of the HepG2 cells transfected with pcDNA-NS5A, and the luciferase activity was up-regulated in the presence of the HCV NS5A protein while the expression of luciferase mRNA showed no difference., Conclusion: HCV NS5A protein can upregulate the HCV IRES activity and this effect is dose-dependent with NS5A.

Objectives: To observe the inhibition of HBV replication and antigen expression by RNA interference aimed at different parts of the HBV genome., Methods: Following the rules of shRNA expression vector design and construction, we constructed seven kinds of sequence specific vectors and two kinds of mutant shRNA expression ones. We then cotransfected those shRNA and HBV expression vectors into HepG2 cells using lipofectamine2000. The level of HBV replication was investigated using Southern blot and the antigen expression using ELISA., Results: The replication of HBV DNA was inhibited by many shRNAs, especially the ones against P1, S2, C2, S1 and X. The inhibition rate against P1 was as high as 95%. Results obtained with ELISA showed that the shRNAs targeting C2, C1 and S2 had high rates of inhibition to HBsAg., Conclusion: The replication and antigen expression of HBV could be inhibited by shRNAs aimed at four different open read frames, and higher inhibition rates of HBV replication and surface antigen expression could be obtained by P1 and C2, respectively.

Objective: To screen and identify proteins that interact with hepatitis C virus NS3 by means of T7-phage display system., Methods: Hepatitis C virus NS3 was expressed by prokaryotic expression and used as a selected molecule to biopan the T7 select human liver cDNA library; the selected positive clones were identified using DNA sequencing and analyzed with BLAST program in GenBank., Results: After BLAST analysis in all the positive clones, the proteins which interacted with the hepatitis C virus NS3 were found to be serpin peptidase inhibitor, clade A, member 1 (SERPINA1) and cyclophilin-LC., Conclusion: T7-phage display system is a convenient, rapid and effective method for screening interacting proteins. The proteins thus selected will provide an important means for studying the pathogenesis and carcinogenesis of HCV.

Objective: To develop a RNAi approach that specifically targets the HCV IRES sequence by vector-expressed short hairpin RNA (shRNA) in vitro, and to assess the inhibitory effect of the shRNA on reporter gene expression., Methods: Eukaryotic expressing plasmids, pIRES-GFP and p5' UTR-Luc containing GFP or luciferase gene controlled by HCV IRES were cotransfected into HepG2 cells with either a RNAi plasmid pshRNA-HCV or a control plasmid pTZU6+1. At 24, 48, 72 hours post transfection, the fluorescence in the transfected cells was studied using fluorescence microscopy. The levels of GFP RNA were determined using RT-PCR and those of protein were determined using Western blot. The activities of luciferase were assayed using a dual luciferase assay system., Results: The introduction of RNAi plasmid efficiently and specifically down-regulated the expression of the reporter gene. RT-PCR showed that the RNAs of GFP gene were distinctly reduced (about 60%) when the pIRES-GFP was cotransfected with pshRNA-HCV, whereas the control vector did not exhibit inhibitory effect on the mRNA level, according to Western blot assay. The luciferase activity also decreased by 60%-70% in comparison to the control plasmid., Conclusion: Our results demonstrate that the shRNA targeting HCV IRES shows a strong inhibitive effect on the expression of the reporter gene controlled by this sequence, suggesting that RNAi-based anti-HCV strategy may represent a potential approach in the therapy of HCV infection.

Objectives: To study the role of histone modification in the regulation of IFN-gamma-activated gene using chromatin immunoprecipitation technique., Methods: Real-time PCR was used to measure the mRNA level of interferon-gamma-inducible protein 10 (IP-10) in Hela cells. Chromatin immunoprecipitation combined with Real-time PCR was used to check the histone H4 acetylation level at IFN-stimulated response element (ISRE) locus of IP-10 gene., Results: IP-10 was strongly activated by IFN-gamma. The histone H4 deacetylation happened at the ISRE locus when IP-10 was induced by IFN-gamma. The activation of IP-10 and the deacetylation of histone H4 at the ISRE site induced by IFN-gamma were inhibited or blocked by histone deacetylase (HDAC) inhibitor trichostatin A (TSA)., Conclusion: The histone H4 deacetylation at the ISRE site is related with the activation of IP-10 by IFN-gamma.

Objective: To investigate the effects of different subtypes IFN alpha (IFN alpha2b, IFN alpha2a, and IFN alpha1b) transduction molecular STAT1, STAT2, IFNAR, PKR, and RNase L, and to study the differences of their antiviral effects and to evaluate the key signaling transduction molecules., Methods: (1) After HepG2 cells were treated with IFN alpha2b, IFN alpha2a, or IFN alpha1b, the mRNA levels of STAT1, STAT2, IFNAR, PKR, and RNase L were detected by RT-PCR. (2) After HepG2 cells were treated with 1000 U/ml IFN alpha2b, IFN alpha2a, or IFN alpha1b, the protein expression levels of STAT1 and IFNAR were examined by Western blot., Results: RT-PCR results: (1) IFNAR, STAT1, and STAT2 mRNA expression levels were slightly higher in the IFN alpha1b group than those in the IFN alpha2b group (P > 0.05). The mRNA expression levels in IFN alpha1b or IFN alpha2b groups were significantly higher than in the IFN alpha2a group (P < 0.05). (2) The PKR mRNA expression showed no significant differences among IFN alpha1b, IFN alpha2b, and IFN alpha2a groups. (3) The RNase L mRNA expression was very weak. We could not compare the differences of the RNase L mRNA levels in different groups by RT-PCR. Western blot results: (1) The IFNAR, and STAT1 protein expressions were greatly up-regulated after IFN alpha induction compared with the untreated group (P < 0.05). (2) The IFNAR, and STAT1 protein expression levels in IFN alpha1b group were slightly higher than the IFN alpha2b group. IFNAR, and STAT1 protein levels of IFN alpha1b or IFN alpha2b group were significantly higher than IFN alpha2a group (P < 0.05)., Conclusion: STAT1, STAT2, IFNAR mRNA and protein expressions could all be markedly up-regulated after IFN alpha treatment. Effects of IFN alpha1b or IFN alpha2b were greatly stronger than IFN alpha2a. The PKR mRNA expression also was greatly up-regulated after IFN alpha treatment. Expression levels of PKR in IFN alpha1b, IFN alpha2b, and IFN alpha2a groups were all similar. The mRNA level results were consistent with the protein level results. Our results showed that the antiviral activity of IFN alpha1b or IFN alpha2b were stronger than that of IFN alpha2a. The signal transduction molecules STAT1, STAT2, and IFNAR could be regarded as a key index to evaluate antiviral activity of IFN alpha. Further confirmation is still needed to see whether PKR could be regarded as a key index.

Objective: To study the promoter activity in HepG2 cells of the DNA sequence corresponding to the HCV 5'UTR., Methods: Plasmids, 5'UTR-Luc(+) and 5'UTR-Luc(-) carrying the forward and reverse DNA sequences corresponding to the HCV 5'UTR respectively were constructed, and subsequently transfected into HepaG2 cells. The luciferase activity and the mRNA of the luciferase gene were then detected. The 5'UTR sequence was cloned into a GFP vector to make 5'UTR-EGFP, and then the GFP expression was confirmed by fluorescence microscopy., Results: 5'UTR-Luc(+) had an obvious luciferase activity whereas 5'UTR-Luc(-) had nearly no luciferase activity. The former had a high level of luciferase mRNA while the latter could not be detected. An intense green fluorescence expression was observed in the cells transfected with the plasmid of 5'UTR-EGFP., Conclusion: The forward DNA sequence corresponding to HCV 5'-UTR had an obvious promoter activity in hepG2 cells. It may play an important role in the replication of HCV.

Objective: To synthesize highly pure HBV post-transcriptional regulatory element (HPRE) via transcription in vitro by T7 RNA polymerase., Methods: HPRE gene was amplified by PCR from a template containing HBV complete genomic DNA and cloned into plasmid pGEM-11zf. The cloned DNA sequence was transcribed by T7 RNA polymerase., Results: The construction of HPRE gene recombinant plasmid and production of HPRE via transcription in vitro was successful., Conclusion: In vitro transcription by T7 RNA polymerase can be used to synthesize highly pure HPRE.

Objective: To detect and compare the transcriptional activities of tumor-specific survivin and AFP promoters in various hepatocellular carcinoma cell lines and to lay some groundwork for targeting gene therapy in human hepatocellular carcinoma., Methods: The fragment of survivin and AFP promoters were acquired by PCR amplification and were cloned into the reporter plasmid pGL3-Basic, which contained a luciferase gene. The constructed eukaryotic expression plasmid pGL3-SUR and pGL3-AFP, in which the expression of the luciferase was derived by survivin or the AFP promoter, were transfected into three HCC cell lines. At 24 hours post transfection (p.t.), the activity of the luciferase was determined with Dual-Luciferase Reporter Assay System. A pGL3-CMV, containing the CMV promoter controlled luciferase gene, was used as a positive control., Results: Both survivin and AFP promoters had transcriptional activities in all three HCC cell lines and the transcriptional activity of the survivin promoter was much higher than the AFP promoter (52-98 times) and reached a level of 16% approximately 21% of the transcriptional activity of the CMV promoter., Conclusion: Our data reveals that the survivin promoter possesses a high transcriptional activity in all three established HCC cell lines and may serve as a useful tool for transcriptional targeting gene therapy of HCCs.

Objective: To investigate the anti-HBV effect of fusion protein thymosin alpha1-interferon alpha (TA1-IFN) in vitro and to compare its effect with a combination of interferon alpha and thymosin alpha1., Methods: After 2.2.15 cells were seeded for 24 hours, drugs of five serial concentrations (8000, 4000, 2000, 1000, 500 U/ml) were added to the wells, then the medium was changed every three days. After 2.2.15 cells were treated with drugs for 6 days, the medium was collected. The inhibitory rates on HBsAg and HBeAg were determined using Abbot kit, and the cytotoxicity of different drugs by means of MTT colorimetric assays was also observed., Results: The inhibitory rate of fusion protein on HBsAg, HBeAg was dose-dependent and reached the maximum at 8000 U/ml concentration. In the meantime, the inhibitory rates of fusion protein on HBsAg and HBeAg were 72.2% +/- 0.8% and 60.4% +/- 1.1% respectively, and the cell survival rate was 85.2% +/- 2.0%; In the corresponding concentration, the inhibitory rates of combination thymosin alpha 1 and interferon alpha on HBsAg and HBeAg were 40.0% +/- 0.7%, 34.5% +/- 3.2% respectively. The results showed significant statistical differences between them; cell survival rate 70.0% +/- 1.9%, and the difference of the results was also significant. Cytotoxicity of fusion protein was weaker than a combination of thymosin alpha 1 and interferon alpha., Conclusion: Fusion protein TA1-IFN exerted stronger anti-HBV effects in vitro. Its anti-HBV effects in vitro were stronger than the combination of thymosin alpha and interferon alpha, and its cytotoxicity was weaker than the combination of thymosin alpha and interferon alpha. Our studies provided important evidence for clinical research on TA1-IFN, and also brought new hope for hepatitis B therapy.

Objectives: By studying the possibility and degree of the replication and expression of human hepatitis B virus (HBV) genes in normal liver cells from heterogenous species, such as primary duck hepatocytes (PDH) and primary rat hepatocytes (PRH), to investigate the species-specificity of HBV infection and replication., Methods: PDH and PRH isolated by in situ perfusion with low concentration collagenase were transfected with complete HBV genome by electroporation (transfection group, about 1.19 10(12) copies of linear HBV DNA/1 10(7) PRH/PDH). From 1 day to 15 days after transfection, HBsAg and HBeAg in the supernatants and lysates of PRH/PDH were measured with IMX System, HBcAg was assayed with western blotting, Immunol dot blotting and Immunocytochemistry. Moreover, HBV S-mRNA and X-mRNA were tested with RT-PCR. Meanwhile, replicative intermediates of HBV DNA were analyzed by Southern blotting and Dot blotting. PRH/PDH electroporated only was used as control group., Results: HBsAg in the lysates of transfected PDH group were 15.24 (1day), 14.55 (3 days) and 5.13 ( 5 days; P/N values, positive>or=2.1), HBeAg all was negative (<2.1), and both were negative in the supernatants of transfected group. Viral antigen production in transfected rat hepatocytes: HBsAg in the lysates of transfected hepatocytes was positive, P/N values ranging from 2.17 to 93.41, The average P/N values was 14.74+/-31.82, and could be maintained for 15 days after transfection. Whereas, HBsAg in the supernatants of transfected group was only found positive on 1 day after transfection, which P/N value was 6.66. HBeAg and HBcAg in the lysates of PRH of transfection group were positive during the first 3 days following transfection, P/N values was around 2.8. The total amount of HBV DNA in the transfected PDH and PRH groups was strongly positive by dot blotting, whereas that of the control group was negative. Southern blot analysis of intracellular total HBV DNA indicated that there were relaxed circular (RC), covalently closed circular (ccc) and single-stranded (SS) HBV DNA replicative intermediates in the transfected PDH and PRH groups, there was no integrated HBV DNA in the cellular genome. Control groups were negative at all., Conclusion: Expression of HBV genes and production can occur in hepatocytes from nonmammalian species or mammalian species, which strongly supports the idea that replication of HBV has no critical species-specificity, and yet it depends on the endoenvironment of hepatocyte.

Objective: To construct the plasmid containing short hairpin RNA (shRNA) of survivin in order to suppress the expression of survivin gene in HepG2 and SMMC-7721., Methods: Two 20 to 21 bp reverse repeated motifs of survivin target sequence with 4 bp or 8 bp spacer were synthesized respectively and inserted into plasmid pTZU6+1 to generate the plasmid pshRNA-survivin1 and pshRNA-survivin2; plasmid pEGFP-C1-survivin and pshRNA-survivin1 or pshRNA-survivin2 plasmid were cotransfected into liver cancer cell HepG2 and SMMC-7721 to detect effect of GFP expression respectively and analyze the inhibition of survivin gene., Results: The recombinant plasmid pshRNA-survivin1 and pshRNA-survivin2 were successfully constructed. The recombinant plasmids suppress the survivin expression by 80% in HepG2 and SMMC-7721., Conclusion: The result showed that the short hairpin RNA of survivin can efficiently suppress it's expression in HepG2 and SMMC-7721.

Objectives: To develop a new method for amplifying and sequencing the full-length of HBV genome., Methods: A pair of primers located at the nick region of HBV molecule and a thermostable polymerase with high fidelity and sensitivity were used. After cloning the PCR products into a plasmid, the sequences of HBV genome were analyzed., Results: The full-length of HBV genome were acquired using this method. The sensitivity and fidelity of the new method were also analyzed. The least quantity of initial templates was 10(2) and the artificial mutation rate was 1.2 bp/kb., Conclusion: This method can be used in amplification and sequence analysis of the full-length of HBV genome on a large scale.

Objectives: To transfer 1.3-fold-overlength genome of HBV expression plasmid into HepG2 cells, and observe the dynamic changes of viral replication as well as expression in the transfected cells., Methods: 4.1 kb fragment of HBV genome, derived from pGEM-HBV, was cloned into Hind III site of the eukaryotic expression vector pCDNA3.1 to construct the recombinant plasmid pHBV. Then HepG2 hepatoma, cells were transfected with pHBV, using Lipofectamine2000 transfection reagent. After 24, 48, 72 hours, the levels of HBsAg and HBeAg in the supernatant of HepG2 cells were determined with Abbott MEIA Kit. Intracellular viral DNA and RNA were analyzed by Southern and northern blot hybridization. In addition, viral-specific proteins (HBsAg and HBcAg) were assayed by immunofluorescence staining., Results: The expression vector pCDNA3.1 was constructed successfully. The levels of HBsAg were 5.36+-0.25, 13.42+-1.24, 7.52+-0.43, and the values of HBeAg were 9.16+-0.32, 22.75+-1.49, 15.96+-1.03 after 24, 48, 72 hours, respectively. All expected HBV replicative intermediates and specific transcripts were verified by Southern and northern blot analysis. The HBsAg-positive cells peaked after 24 hours, and then dropped slowly. HBsAg positive staining scattered in the cytoplasm, whereas HBcAg lied maily in the cytoplasm apart from nuclears., Conclusions: This recombinant plasmid, which initiates viral replication efficiently in infected cells, is expected as a novel tool for investigating HBV replication in vitro.

Objective: To clone and analyze duck hepatitis B virus genome from Chongqing brown duck., Methods: Duck hepatitis B virus (DHBV) DNA extracted from a Chongqing brown duck was amplified by PCR and cloned into PGEM-T vector using T-A clone method. The sequence of this DHBV genome was analyzed with some softwares after identified., Results: The duck hepatitis B virus genome from Chongqing brown duck (DHBVcq), which was 3 024 nucleotides long, contained three ORFs whose onset and end nucleotides were in accord with those of HPUGA, encoding P, PreC/C and PreS/S protein respectively. Comparison of this strain with other DHBV reported in GenBank showed that the homology of DHBVcq and M32990 got the highest score of 94.9% at nucleotide level, while DHBVcq and DHBVCG got the least (89.8%). Most of the conserved regulation nucleotides and amino acids sequence found in other DHBV were also identified in DHBVcq. The epsilon region of DHBVcq, which was important for encapsidation of pgRNA and synthesis of minus-strand DNA, differed from that of most other DHBV strains, forming a stem-loop conformation with a three- nucleotides upper stem rather than a common nine-nucleotides one in free status., Conclusion: The successful clone and analysis of DHBVcq provide further studies with helpful information.

Objective: To evaluate their long-term outcome and the efficacy and economic significance of antiviral drugs by investigating the long-term health-related quality of life (HQL) in chronic hepatitis B (CHB) patients., Methods: The HQL of 101 CHB patients with biopsy-proven 6 to 18 years ago and 105 persons of general population as control was studied with revised SF-36 questionnaire., Results: The HQL in CHB patients was lower than that in general population in physical functioning, role physical, general health, mental health, and specific symptoms (mu > or = 2.10, P<0.05)., Conclusions: The long-term HQL in chronic hepatitis B patients is poor.

Objective: To establish an in vitro model of hepatitis B virus (HBV) replication and expression in primary rat hepatocytes (PRH) transfected with circular viral DNA for further study on the interaction of HBV with hepatocytes., Methods: Circular viral DNA containing complete HBV genome were transfected into PRH by electroporation (transfected group, about 4mug of circular viral DNA/1 10(7)cells). From day 1 to day 10 after transfection, HBsAg and HBeAg in the supernatants and lysates of PRH were measured with IMX system. HBcAg was assayed with western blotting, immunol dot blotting and immunocytochemistry. Meanwhile, HBV S-mRNA and X-mRNA were tested with RT-PCR, and replicative intermediates of HBV DNA were analyzed by southern blotting and dot blotting. Moreover, Transmission electron microscopy was used if viral particles were produced in transfected rat hepatocytes. PRH electroporated only was used as control group., Results: (1) Viral antigen production in transfected rat hepatocytes: HBsAg in cell lysates was positive. P/N values ranged from 4.83 to 85.69, and could be maintained for 10 days after transfection. The average P/N values was 18.239 27.459. Whereas, HBsAg was negative in the supernatants of transfected group (P/N values, negative<2.1). HBeAg in the supernatants and lysates of transfected hepatocytes all was negative (P/N values<2.1) during 10 days following transfection. HBcAg was only found positive in transfected hepatocytes by immunol dot blotting. (2) Detection of viral transcripts: transcription of HBV DNA was investigated by preparing total RNA from rat hepatocytes 2 days after transfection and looking for S-mRNA and X-mRNA by RT-PCR. Results showed S-mRNA positive, X-mRNA negative. (3) HBV DNA replication analysis: intracellular total DNA was extracted 2 days after transfection and analysed by southern blotting. All replicative DNA intermediates, including relaxed circular (rcDNA), covalently closed circular (cccDNA), and single-stranded (ssDNA) linear HBV DNA forms, were indicated. Dot blotting showed intracellular HBV DNA positive in transfected group during 10 days after transfection. However, viral particles were not found in transfected hepatocytes during 3 days after transfection., Conclusions: Circular HBV DNA transfected into primary adult rat hepatocytes could obtain continuous replication and stable expression of HBV surface antigen. This in vitro model has high reproducibility and stability, and is useful for directly studying the interaction of HBV with hepatocytes.