S gene mutations in HBsAg/HBsAb double positive chronic hepatitis B patients / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To investigate S gene mutations in HBsAg/HBsAb double positive chronic hepatitis B patients.</p><p><b>METHODS</b>HBV S gene from 8 patients (Group A) with HBsAg (+)/HBsAb (+) and 9 patients (Group B) with HBsAg (+)/HBsAb (-)was amplified by polymerase chain reaction (PCR) and sequencing. Both the distribution of genotype and serotype and the rate of MHR region were compared by Fisher's exact test. The mutation rate of both the DNA level and amino acid level was compared by t test.</p><p><b>RESULTS</b>No significant difference in distribution of genotypes was found between the two groups (P=0.153). In group A, 2 were genotype B, 6 were genotype C; In group B, 6 were genotype B, 3 were genotype C. No significant difference in distribution of serotypes was found between the two groups, either (P=0.218). In group A, 2 were adw, 5 were adr, 1 was ayr; In group B, 6 were adw, 3 were adr. The mutation rate of Pre-S1 region at both the DNA level (2.29% vs 1.80%, t=2.66, P more than 0.05) and the amino acid level (2.66% vs 1.59%, t=1.39, P>0.05) was not significantly different between these two groups; the mutation rate of Pre-S2 region in group A patients was significantly higher than that in group B at the DNA level (1.74% vs 0.91%, t=4.68, P<0.01), but not higher at the amino acid level (3.18% vs 2.05%, t=1.85, P>0.05), the mutation rate of S region in group A patients was significantly higher than that in group B at both the DNA level (2.13% vs 0.81%, t=6.00, P<0.01) and the amino acid level (4.37% vs 1.52%, t=5.32, P<0.01). Amino acid substitutions were found both within and beyond the MHR region. The rate of "a" determinant mutations in these two groups was also found to be significantly different (P<0.05).</p><p><b>CONCLUSION</b>Higher HBV S gene mutation rate exists in HBsAg/HBsAb double positive patients than that in HBsAg (+)/HBsAb (-) patients.</p>

The immunization effects of HBV core antigen and surface antigen fusion protein in mice / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To evaluate the immunization effects of HBV core antigen and surface antigen fusion protein.</p><p><b>METHODS</b>The DNA fragments encoding HBsAg 100-162 aa; HBcAg 1-78 aa and HBcAg 83-144 aa were PCR-amplified, and then cloned into pcDNA3 plasmid. The chimeric gene was subcloned into the prokaryotic vector, pRSET-B. The E.coli expressed recombinant protein purified. BALB/c mice were immunized with recombinant protein or eukaryotic expression plasmid, humoral response and cellular response were examined.</p><p><b>RESULTS</b>The plasmid containing the chimeric gene of HBsAg and HBcAg induced effective anti-HBs antibody response and strong HBcAg specific lymphocyte proliferative response, but could not induce anti-HBc antibody response. Fusion protein induced strong anti-HBs and anti-HBc antibody response, and it also caused significant HBcAg specific lymphocyte proliferation. Compared to the recombinant fusion protein, the plasmid containing the chimeric gene of HBsAg and HBcAg can induce more effective cellular response but weaker humoral response.</p><p><b>CONCLUSION</b>Compared to the recombinant fusion protein, the plasmid containing the chimeric gene of HBsAg and HBcAg is a more effective vaccine.</p>

Expressions of phosphorylated-Smad2 and PTEN in hepatocellular carcinomas and adjacent liver tissues / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the expressions of phosphorylated Smad2 (P-Smad2) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) in hepatocellular carcinoma (HCC) tissues.</p><p><b>METHODS</b>The expressions of P-Smad2 and PTEN were detected using Envision immunohistochemical technique in 31 cases of HCC tissues, 25 cases of HCC adjacent liver tissues and 13 cases of non-hepatocellular carcinoma tissues.</p><p><b>RESULTS</b>The positive expression and staining intensity of PTEN in the cytoplasm of HCC cells (64.5%, 4.19+/-3.31) was significantly lower than those of the cells of the cancer adjacent tissues and non-cancerous tissues (96.0%, 7.88+/-0.93; 100%, 7.77+/-0.93). The staining intensity of PTEN in the cytoplasm of Edmondson pathologic grade III HCC cells was lower than those of the Edmondson grade I. The expression of PTEN was negatively correlated with intrahepatic vascular cancer thrombi (r=-0.43) and the expression of PTEN in the nuclei or cytoplasm of liver cells was negatively correlated with the liver disease progressions (r=-0.34). The positive rate and expression intensity of phosphorylated Smad2 in nuclei of HCC cells were the same as those in cancer adjacent and non-tumor liver tissues. The expression was mostly in the nucleus and cytoplasm of Edmondson grade I HCC cells, cancer adjacent liver tissue cells and non-tumor liver tissues, but its expression was only in the nuclei of Edmondson grade II and III HCC cells. The phosphorylated Smad2 expression appeared in the nuclei and in the cytoplasm of liver cells and it was positively correlated with the severity of the tumor pathology (r=0.22). Spearman correlation analysis revealed a significant inverse correlation between PTEN and phosphorylated Smad2 in HCC tissues (r=-0.73).</p><p><b>CONCLUSIONS</b>The aberrant expressions of PTEN and phosphorylated Smad2 and their interaction may play an important role in the pathogenesis of hepatocellular carcinoma.</p>

Common linear B-cell epitopes in human hepatitis B virus core protein and woodchuck hepatitis virus core protein / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To search for and verify some common B cell epitopes in the core proteins of woodchuck hepatitis virus and human hepatitis B virus.</p><p><b>METHODS</b>Monoclonal 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.</p><p><b>RESULTS</b>Cross-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.</p><p><b>CONCLUSION</b>The 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.</p>

Antigenicity of major hydrophilic region II of hepatitis B virus surface antigen / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study whether the substitutions at the major hydrophilic region II (MHRII) of hepatitis B surface antigen (HBsAg) will impair the antigenicity of HBsAg.</p><p><b>METHODS</b>Four recombinant plasmids expressing mutant HBsAg (mtHBsAg) P1120T, C121S, K122I and T123N were constructed. HepG2 cells were transfected with the four plasmids and a plasmid expressing G145R HBsAg. The immunoreactivity of the cells expressing mtHBsAg with P1120T, C121S, K122I, T123N and G145R were detected by immunofluorescence (IF) staining and ELISA with 4 antibodies and 7 HBsAg diagnostic kits respectively.</p><p><b>RESULTS</b>mtHBsAg with P120T was recognized by mAb1 and mAb2. mtHBsAg with C121S and K122I was not recognized by any mAbs. mtHBsAg with T123N in lysates was recognized by mAb2, but not recognized in the supernatants.</p><p><b>CONCLUSION</b>Substitutions at amino acid positions 120-123 of HBsAg strongly impaired the antigenicity of HBsAg, a fact that was not appreciated previously.</p>

The growth inhibition effects of TSLC1 gene on human hepatocyte carcinoma cell line HepG2 / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To study the effects of tumor suppressor in lung cancer-1 (TSLC1) on human hepatocyte carcinoma cell line HepG2.</p><p><b>METHODS</b>A full length of TSLC1 cDNA was amplified from RNA of normal human liver cells by RT-PCR, and it was cloned into a pCI-neo expression vector and transfected into human hepatocellular carcinoma cell line HepG2. The HepG2 cells transfected with this plasmid (experimental group) and those treated with pCI-neo vector (control group) and without any treatment (blank group) were compared. Cell morphology was studied microscopically and cell growth was analyzed with MTT assay. FACSort flow cytometry analysis was performed to assess the cell cycle distribution and apoptosis.</p><p><b>RESULTS</b>A stable cell line expressing TSLC1 protein was successfully established. Morphologically, cells of the experimental group were tightly aggregated when compared with those of the control and blank groups. The growth of TSLC1-transfected cells was significantly suppressed in vitro compared with those of the control and blank groups. The amount of G0-G1 cells was 63.66%+/-3.83% (P less than 0.01) in the experimental group, while those of the control and blank groups were 47.45%+/-0.91% and 54.47%+/-0.96% respectively. The amount of S phase cells in the experimental group, 22.90%+/-6.04%, was significantly lower (P less than 0.05) than that of the control group (36.58%+/-0.61%) and the blank group (33.61%+/-2.99%), which suggested a G0-G1 cell cycle arresting. The number of cells in early and late phase apoptosis (17.09%+/-0.20% and 16.11%+/-0.40% respectively) were significantly higher than those of the control and blank groups (P less than 0.01).</p><p><b>CONCLUSIONS</b>TSLC1 strongly inhibits the growth of HepG2 cells in vitro and induces apoptosis of the cells, suggesting that TSLC1 may have a tumor suppressor function in HCC.</p>

Inhibition of hepatitis B and duck hepatitis B virus replication by APOBEC3G / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) mediated antiviral activity against hepatitis B virus (HBV) and duck hepatitis B virus (DHBV).</p><p><b>METHODS</b>Total RNA was extracted from peripheral blood mononuclear cells (PBMCs), RT-PCR product was cloned into the EcoR I/Hind III restriction sites of the CMV-driven expression vector fused with a hemagglutinin fusion epitope tag at its carboxyl terminal. Replication competent 1.3 fold over-length HBV was constructed with full-length HBV of ayw subtype. The mammalian hepatoma cell HepG2 was cotransfected with the replication competent 1.3 fold over-length HBV and various amounts of CMV-driven expression vector encoding APOBEC3G-HA. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA, HBV DNA. RNA from intracellular core particles was examined using Northern and Southern blot analyses. Chicken hepatoma cell LMH was cotransfected with head-to-tail dimer of an EcoR I monomer of DHBV and various amounts of CMV-driven expression vector encoding APOBEC3G-HA. DHBV DNA from intracellular core particles was examined using Southern blot analysis.</p><p><b>RESULTS</b>CMV-driven expression vector encoding APOBEC3G-HA and replication competent 1.3 fold over-length HBV were constructed. There was a dose dependent decrease in the levels of intracellular core-associated viral (HBV and DHBV) DNA and extracellular production of HBsAg and HBeAg. Levels of intracellular core-associated viral RNA were also decreased, but the expression of HBcAg remained almost unchanged.</p><p><b>CONCLUSION</b>APOBEC3G suppresses HBV and DHBV replication and also suppresses HBsAg and HBeAg expression.</p>

Cloning, prokaryotic expression and polyclonal antibody preparation of HCCR: a new biomarker for hepatocellular carcinoma / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To construct a prokaryotic plasmid expressing truncated human cervical cancer oncogene (HCCR-1(167-360)), to express and purify the recombinant protein, and to develop the polyclonal antibody against HCCR.</p><p><b>METHODS</b>HCCR-1(167-360) was amplified by RT-PCR from HepG2 cells and cloned into vector pRSET-B, then expressed in E.coli BL21(DE3) pLysS, which was induced by IPTG. The recombinant protein was purified using Ni-NTA spin column and acrylamide gel electrophoresis. A polyclonal antibody was developed by immunizing BALB/c mice with the purified recombinant protein, and their sensitivity and specificity were tested using enzyme-linked immunosorbent assay, immunohistochemical staining and Western blot analysis.</p><p><b>RESULTS</b>Recombinant plasmid expressing truncated HCCR-1167-360 was constructed. A protein of 2.70 x 10(4) was successfully expressed and purified. High titer polyclonal antibody with a high specificity was obtained by immunizing BALB/c mice with the purified recombinant protein.</p><p><b>CONCLUSIONS</b>The truncated recombinant HCCR-1(167-360) developed in this study is highly purified and shows strong antigenecity; the polyclonal antibody against this HCCR protein was generated by regular immunization method, showing both high sensitivity and specificity. The protein and the antibody can be used for further clinical examination and research of HCCR.</p>

Expression of interferon alpha family gene of Chinese marmot in eukaryotic and prokaryotic cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the function of interferon alpha (IFNalpha) in a Chinese marmot model of hepatitis B, we expressed the Chinese marmot (Marmota himalayana) IFNalpha family gene (IFNA) in eukaryotic cells and prokaryotic cells.</p><p><b>METHODS</b>Eukaryotic and prokaryotic expression plasmids harboring Chinese marmot interferon alpha gene with different genotypes were generated using molecular cloning technology. We detected the biological activity of all expression products by viral protection assay, and analyzed their differences and species restriction of the biological activity.</p><p><b>RESULTS</b>The Chinese marmot functional genotype IFNalpha was expressed in the baby hamster kidney (BHK) cell line, and these products protected WH12/6 cells challenged by encephalomyocarditis virus (EMCV). The Chinese marmot IFN-alpha5 also expressed in E. Coli induced by IPTG, and purified fusion protein had antiviral biological activity. The biologic activity displayed differences among different subtype IFNalpha, and it had strict species restriction.</p><p><b>CONCLUSION</b>The IFNalpha family gene of the Chinese marmot can be expressed in both eukaryotic and prokaryotic cells, and the expression products show antiviral activity in a protection assay. This study provides, for the first time, evidence that IFNalpha from the Chinese marmot has an antiviral function in vitro and can be used to improve the efficacy of current therapies for HBV infection in our Chinese marmot model.</p>

Distribution of hepatitis B virus genotypes in Hubei province and its clinical significance / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the distribution of hepatitis B virus genotype in Hubei province (China) and its clinical significance.</p><p><b>METHODS</b>Serum samples from 190 HBV DNA positive patients with chronic HBV infection,including 52 asymptomatic HBV carriers (ASC), 56 chronic hepatitis (CH), 32 fulminant hepatic failure (FHF), 22 liver cirrhosis (LC), and 28 hepatocellular carcinoma (HCC) patients were collected and tested for HBV genotypes by type-specific primers.</p><p><b>RESULTS</b>A simple and precise genotyping system based on PCR using type-specific primers was developed for the determination of genotypes of hepatitis B virus (HBV). Of the 190 patients, 140 (73.7%) were genotype B and 42 (22.1%) were genotype C. Genotype B was more prevalent in the FHF and HCC patients than in the ASC patients; the ALT value was significantly higher in genotype B than in genotype C patients. The rate of anti-HBe was significantly higher in genotype B than in genotype C except in the patients of the ASC group.</p><p><b>CONCLUSION</b>The system we used seems to be a useful tool for the molecular diagnosis of HBV infection and for large-scale surveys. Genotype B, genotype C and BC combination exist in Hubei province, and genotype B is the major genotype in this area especially in FHF and HCC patients.</p>