Meta-analysis of magnesium isoglycyrrhizinate combined with nucleoside analogues in patients with chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To evaluate the efficacy and safety of the magnesium isoglycyrrhizinate plus nucleoside analogues (MGL + NA) combination therapy in patients with chronic hepatitis B using a meta-analysis approach.</p><p><b>METHODS</b>The Chinese Biochemical literature on Disc (CBMDisc) and the Chinese Scientific Journal database, CNKI, were searched for randomized controlled trials (RCTs) of MGL+NA in patients with chronic hepatitis B published between 1995 and 2013. Data related to treatment type (combination therapy vs. mono-therapy) and outcome (markers of efficacy and safety, including levels of hepatitis B virus (HBV) DNA, hepatitis B e antigen (HBeAg), alanine aminotransferase (ALT) and aspartate aminotransferase (AST)). Weighted mean differences (WMD) were calculated and the Peto method was used to determine the relative risk (RR), both with 95% confidence intervals (CIs).</p><p><b>RESULTS</b>Meta-analysis of the six included RCTs of MGL + NA, representing a 704 patients with chronic hepatitis B, showed WMDs for ALT of -12.98 (95% CI: -18.24 to -7.71, P less than 0.01) and for AST of -9.49 (95% CI: -14.53 to -4.45, P = 0.0002) and RRs for HBeAg of 1.79 (95% CI: 1.17 to 2.76, P = 0.008) and for HBV DNA of 1.35 (95% CI: 1.05 to 1.74, P = 0.02). The therapeutic efficacy of MGL+NA combination therapy was better than that of NA mono-therapy (P less than 0.01).</p><p><b>CONCLUSION</b>For patients with chronic hepatitis B, MGL combination therapy may enhance the antiviral efficacy of NA treatment and help to improve liver function during treatment.</p>

Characteristics of HBcAg(18-27) CTL epitopes of the main epidemic HBV strains in China / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the characteristics of the virology background of HLA-A2 restricted HBcAg(18-27) epitope mutations in HBV infected patients in China.</p><p><b>METHOD</b>30 HBV sequences with different genotypes from Genbank were analyzed by bioinformatics and the mismatched primers were designed for constructing a PCR-RFLP method to screen HBcAg(18-27)V/I in China. The distributions of HBcAg(18-27)V/I of 160 samples with HBV genotype B/C infection from 8 areas in China were screened and analyzed by PCR-RFLP and sequencing. The affinity of HBcAg(18-27)V/I to HLA-A0201 was analyzed through referencing the bioinformatics websites.</p><p><b>RESULTS</b>We successfully constructed a PCR-RFLP method for screening HBcAg(18-27)V/I from genotype B/C, and only 3 samples with HBcAg(18-27)V sequence were found in the 160 samples (3/160, 1.88%). The affinity of HBcAg(18-27)I to HLA-A 0201 was lower than the one of HBcAg(18-27)V through bioinformatic analysis (HLA ligand score was 123 vs 156, and the SYFPEITHI score was 22 vs 24).</p><p><b>CONCLUSION</b>The last amino acid of most HBcAg(18-27) sequences of epidemic HBV strains in China is isoleucine, and not valine. Therefore HBcAg(18-27) sequence background in different HBV genotypes should be thoroughly considered when using it as a reference or control in immunological research about HBV.</p>

Analysis of T cell receptor BV dominant usage and CDR3 sequences during acute exacerbation in patients with chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To understand the role cellular immunology plays in the pathogenesis of chronic hepatitis B (CHB) through analysis of T cell receptor (TCR) beta chain variable region gene (BV) family dominant usage and beta chain complementarity determining region3 (CDR3) sequences of peripheral blood mononuclear cells of the patients.</p><p><b>METHODS</b>TCR BV families were amplified by inverse polymerase chain reaction (RT-PCR), and the dominant usage of BV families and CDR3 repertoire were analyzed by immunoscope technology for 8 CHB patients during their acute exacerbations and for 4 healthy blood donors who served as controls. The clonality of the T cells suspected by immunoscope was further confirmed by CDR3 sequencing.</p><p><b>RESULTS</b>The TCR BV CDR3 repertoire of the 4 healthy blood donors showed a Gaussian distribution. In the 8 CHB patients, however, the clonal expansion of T cells showed different TCR BV families with each patient. The T cells of the clonal expansion shared different CDR3 sequences.</p><p><b>CONCLUSION</b>The peripheral blood T cells of CHB patients during their acute exacerbation showed significantly a clonal expansion and their T cell clonal expansion may be stimulated by several HBV epitopes. These results indicate that cellular immunology is involved in the pathogenesis of the liver inflammation process of CHB.</p>

Inhibitory effect of siRNA on HPA gene expression in SKOV3 cells / 中国肿瘤生物治疗杂志

Objective:To study the inhibitory effect of siRNA on Heparanase (HPA) expression in SKOV3 cells. Methods:Two pairs of 21 bp reverse repeated sequence targeting HPA RNA (spaced by 9 bp nueleotide) were synthe- sized and were cloned into plasmid PGenesil-1 to construct recombinant plasmid PGenesil-1(+)-HPA expressing 2 hair- pin siRNAs.The inhibition of HPA gene was detected by RT-PCR,real-time PCR and immunohistochemical staining after PGenesil-1(+)-HPA was stably transfected into SKOV3 cells.Results:The recombinant plasmid PGenesil-1(+)-HPA (expressing 2 hairpin siRNAs) was successfully constructed.RT-PCR,real-time PCR and immunohistochemical staining showed that there was a significant decrease in HPA mRNA and protein level in experimental group compared with those in control group.Conclusion:siRNA targeting HPA mRNA can specically suppress the expression of HPA gene in SKOV3 cells;RNA interference method provides a new way for studying the role of HPA and gene therapy of cancer.

Activity of specific deoxyribozymes to cleave hepatitis C virus RNA in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To analyze the cleavage activity of two deoxyribozymes targeting at hepatitis C virus (HCV) RNA in vitro and evaluate their prospects of antiviral therapy.</p><p><b>METHODS</b>Two specific sequences containing 5' ...A / U... 3' in HCV 5'-noncoding region and 5'-fragment of C region (5'-NCR-C) were selected as the target sites, and with the active region of 5'GGCTAGCTACAACGA3', two phosphorothioate deoxyribozymes (TDRz) named as TDRz-127 and TDRz1 were synthesized. HCV RNA 5'-NCR-C was transcribed in vitro from plasmid pHCV-neo which was completely linearized with restriction endonuclease Nar I, and its 5'-end phosphoric acid was deleted by calf intestinal alkaline phosphatase (CIP), then radiolabelled with T4 polynucleotide kinase and gamma-32P-ATP. Under the conditions such as pH 7.5 and a 10 mmol/L Mg2+ concentration, TDRz-127 and TDRz1 were separately (a 5 micromol/L final concentration) or combinedly (each 2.5 micromol/L) mixed with the substrate RNA (200 nmol/L). After denaturation and then renaturation, the reaction systems were incubated in 37 degrees C, and aliquots were removed to terminate the reaction at intended time points. The cleavage products were separated with 8% denaturated polyacrylamide gel electrophoresis and displayed by autoradiography. Finally, the optical density of each product band was measured with Gel Documentation-Analyzing Systems for calculating the percentages of cleaved HCV 5'-NCR-C.</p><p><b>RESULTS</b>After reaction for 15, 30, 45, 60, 75 and 90 min under the adopted conditions, about 8.3%, 16.1%, 24.3%, 26.2%, 29.4% and 31.1% of HCV 5'-NCR-C was cleaved by TDRz-127 respectively; 7.4%, 13.0%, 15.6%, 18.7%, 19.4% and 20.3% by TDRz1; and 15.1%, 29.6%, 37.8%, 39.1%, 41.5%, 42.6% by combining the two TDRzs.</p><p><b>CONCLUSIONS</b>Cleavage percentage of both TDRz-127 and TDRz1 increases with the time, and the effect of combining the two TDRzs is better than that of anyone.</p>

Design and activity assessment of HDV ribozymes to trans-cleave HCV RNA / 第三军医大学学报

Objective　To study the probability of using hepatitis D virus (HDV) ribozyme as a kind of anti-hepatitis-C-virus (HCV) gene thera-py drugs. Methods　The natural HDV genomic ribozyme′s stem Ⅳ was optimized and its substrate-binding region reconstructed, thus three recombinant HCV-specific HDV genomic ribozymes RzC1, RzC2 and RzC3 were obtained. HCV RNA 5'-noncoding region and 5'-fragment of C region (HCV RNA5'-NCR-C) were transcribed from plasmid pHCV-neo by T7 phage RNA polymerase in vitro, and radiolabelled at its 5'-end. The trans-cleaving reaction was performed by mixing the ribozymes and substrate at mol ratio 100∶1 under conditions as follows: 37℃, pH7.5, Mg2+ 20 mmol/L and deionized formamide 2.5 mol/L. Percentage of trans-cleaved products were calculated at different time points and used as the activity indicator of the three ribozymes. Results　RzC1, RzC2 trans-cleaved more substrate when the time extended, and got to 24.9%,20.3% after reac-ting for 90 minutes respectively; RzC3 was not able to trans-cleave its substrate. Conclusion　Recombinant HDV genomic ribozymes have the ability to trans-cleave HCV RNA, but the appropriate target sequence should be selected.

Design and activity assessment of HDV ribozymes to trans-cleave HCV RNA / 第三军医大学学报

Objective　To study the probability of using hepatitis D virus (HDV) ribozyme as a kind of anti-hepatitis-C-virus (HCV) gene thera-py drugs. Methods　The natural HDV genomic ribozyme′s stem Ⅳ was optimized and its substrate-binding region reconstructed, thus three recombinant HCV-specific HDV genomic ribozymes RzC1, RzC2 and RzC3 were obtained. HCV RNA 5'-noncoding region and 5'-fragment of C region (HCV RNA5'-NCR-C) were transcribed from plasmid pHCV-neo by T7 phage RNA polymerase in vitro, and radiolabelled at its 5'-end. The trans-cleaving reaction was performed by mixing the ribozymes and substrate at mol ratio 100∶1 under conditions as follows: 37℃, pH7.5, Mg2+ 20 mmol/L and deionized formamide 2.5 mol/L. Percentage of trans-cleaved products were calculated at different time points and used as the activity indicator of the three ribozymes. Results　RzC1, RzC2 trans-cleaved more substrate when the time extended, and got to 24.9%,20.3% after reac-ting for 90 minutes respectively; RzC3 was not able to trans-cleave its substrate. Conclusion　Recombinant HDV genomic ribozymes have the ability to trans-cleave HCV RNA, but the appropriate target sequence should be selected.