Retroviral vector-mediated HBsAg expression and its stability / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To explore use of retroviral vector in gene therapy of hepatitis B.</p><p><b>METHODS</b>The recombinant vector Plxsn-HBs was constructed by inserting HBV S gene into pLXSN. The pseudovirus, which was produced from PA317 after transferring with pLXSN-HBs by electroporation, were frozen at different temperature. The activities of the pseudovirus to infect eukaryotic cells and express antigen were determined by comparing the numbers of G418-resistant clones and assaying HBsAg in the supernatant of the cells with ELISA after infection HepG2, NIH3T3 and 293 cells.</p><p><b>RESULTS</b>It was hard to find changes in HBsAg amount at different intervals and different temperatures. G418 resistant clones, however, were variable. When frozen at -20 degrees C, the numbers of clones were half less than that of the beginning after 6 months, few clones were formed after 12 months, and no clone was found after 24 months. When frozen at -40 degrees C, the numbers of clones were 121, 332 and 89 42, 137 and 43 for HepG2, NIH3T3 and 293 cell lines at 12 and 24 months, respectively. When frozen at -70 degrees C, the numbers of clones were 159 463 and 112 for HepG2, NIH3T3 and 293 cell lines at 24 months, respectively. There was no statistical difference compared to that of zero months.</p><p><b>CONCLUSIONS</b>The activity of the peseudovirus to infect eukaryotic cells and expressed antigen was not changed after 2 years frozen at -70 degrees C.</p>

An extended two-year trial of lamivudine in Chinese patients with chronic hepatitis B / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To evaluate the long-term efficacy and safety of lamivudine therapy for the treatment of chronic hepatitis B and the clinical influence of emergence of tyrosine methionine aspartic acid (YMDD) motif mutation of hepatitis B virus (HBV).</p><p><b>METHODS</b>This multicenter, double-blind, randomized, placebo controlled trial began in 1996. A total of 429 patients with HBsAg, HBeAg and HBV CNA positives were enrolled. They were randomized to receive either lamivudine 100 mg daily (n = 322) or placebo (n = 107) on 3 : 1 ratio for the first 12 weeks. Thereafter all patients were offered open label lamivudine treatment and assessed every 4 weeks for a total of 104 weeks.</p><p><b>RESULTS</b>After 1 year treatment 72.7% patients (285/392) had a sustained serum HBV DNA response. HBV DNA continued to be substantially suppressed at the second year, except in patients with the emergence of YMDD mutation whose mean HBV DNA levels increased to 86 Meq/ml (bDNA assay) but were much more lower than that of pre-treatment baseline level. lamivudine therapy resulted in increased HBeAg loss and HBeAg/anti-HBe seroconversion, which were correlated with both baseline alanine transaminase (ALT) levels and also with duration of lamivudine treatment. HBeAg loss was achieved in 26.8% of patients with ALT > 1-fold upper limit of normal at 2 yeas and in 35.6% and 55.6% of patients with ALT > 2-fold upper limit of normal and ALT > 5-fold upper limit of normal, respectively. For HBeAg seroconversion, these figures were 17.4%, 22.2%, and 33.3% respectively. By the end of 2 years, ALT levels were remained in normal ranges in 50.3% whose ALT were abnormal before treatment, and in 83% whose ALT were mormal before treatment. YMDD mutation were developed in 49.7% of the patients. Their serum HBV DNA levels were slightly increased to bDNA median level 86 Meq/ml and 15% of the patients they were ALT exceeded baseline levels. Four patients clinically flared-up and recovered after stop treatment. The adverse drug reactions (ADRs) of lamivudine were mild to moderate, only two patients were reported as drug related severe ADR.</p><p><b>CONCLUSION</b>Sustained HBV replication and clinical improvement could be obtained by the long-term lamivudine therapy with good tolerance and safety.</p>

Construction of c-myb Antisense RNA Recombinant Retroviral Vector and Establishment of Its Packaging Cell Line PA317 / 中山大学学报(医学科学版)

【Objective】To construct the c-myb antisense RNA recombinant retroviral vector and its packaging cell line.【Methods】The segment of c-myb gene was cloned into pUC19 with TA cloning method after amplication by RT-PCR,and then was subcloned into retroviral vector pDOR.The recombinant retroviral vector named pDOR-myb was transfected into retroviral package cell line PA317 after selection with G418.【Results】Sequencing data indicated that the c-myb gene was exactly identical to the sequence in the GenBank.The segment of c-myb gene was inserted directionally into pDOR.Resistant colonies were obtained and the titers of pDOR-myb were 5.2×104～9.5×104 CFU/mL.【Conclusion】The recombinant retroviral vector containing c-myb gene is successfully constructed and its packaging cell line PA317/pDOR-myb was established.

Construction of human anti-HBc ScFv eukaryotic expression vector and expression of anti-HBc ScFv in HepG2 cells / 中国病理生理杂志

AIM: To construct an eukaryotic expression vector of human single-chain variable fragment ~against hepatitis B virus core protein (anti-HBc ScFv) and detect its expression in HepG2 cells. METHODS: Anti-HBc ScFv genes were amplified from the plasmids abstracted from positive clone and inserted into pEGFP-c1 vector that contained green fluorescent protein gene. The recombinant plasmids were transfected into HepG2 cells, and resistant clones were obtained by G418 selection. The expression of the gene of fusion protein was determined by fluorescent invert microscope and ELISA. RESULTS: Recombinant plasmids were successfully constructed. The plasmid transfected HepG2 cells were obtained by G418 selection. Specific fluorescence was observed in HepG2 cells 48 hours after transfection. ELISA analysis confirmed the expression of anti-HBc ScFv in the cells. CONCLUSION: The construction of human anti-HBc ScFv eukaryotic expression vector and its expression in HepG2 cells lay the foundation for advanced research of intracellular anti-HBc ScFv.

Investigation on the function and morphometrics of cultured primary rat ~hepatocytes / 中国病理生理杂志

AIM: To investigate the function and morphological changes of long-term cultured primary rat hepatocytes. METHODS: Rat primary hepatocytes were isolated by two-step in situ collagenase perfusion method, and then were purified by density and grade centrifugal method with Percoll. Cell viability was observed by 0.4% trypan blue. The hepatocytes were seeded into 6 wells plate with HepatoZYME-SFM medium. ALT, AST, albumin and urea levels in the supernatant were measured, CYPⅠA1 was detected with EROD method. RESULTS: (2-3)?108 cells per whole liver were obtained with viability and purity above 90% after purified with Percoll. Hepatocytes cultured in HepatoZYME-SFM grew well with normal hepatocyte morphometrics. ALT, AST levels in the supernatant decreased after 3-day culture, and kept at a stable level after 6-9 days. Albumin secretion and urea synthesis were maintained at high levels in 18 days, while CYPⅠA 1 enzyme activity was only detected in 3-6 days. CONCLUSIONS: Percoll was used to increase the viability and purity of freshly isolated rat hepatocytes. Hepatocyte morphometrics and their biological synthesized function are effectively maintained in HepatoZYME-SFM medium.

Effects of c-myb antisense RNA on the proliferation and collagen Ⅰ gene expression in cultured hepatic stellate cells / 中国病理生理杂志

98%. In the pDOR-myb infected HSCs, c-myb expression levels, the cell proliferation, and ? 1-Ⅰ collagen mRNA expression were repressed significantly. CONCLUSIONS: c-myb plays a key role in the activation and proliferation of HSC. c-myb antisense RNA can inhibit cell proliferation and ? 1-Ⅰ collagen mRNA expression in the infected HSC. These data suggest that inhibition of c-myb gene expression would be a potential way for the treatment of liver fibrosis.

Screening,identifying and sequencing of human single-chain variable fragment specific to hepatitis B virus core protein / 中国病理生理杂志

AIM: To Screen and identify human single-chain variable fragment (ScFv) specific to hepatitis B virus core protein and determine its gene sequence. METHODS: The recombinant phages were panned by HBcAg coated in a 96-pore plate and 48 clones were identified specific to HBc after three rounds of panning. The specificity of ScFv from the positive clone was determined by ELISA. Then, the soluble ScFv was expressed in E.coli. HB2151 and secreted in the supernatant. Subsequently, SDS-PAGE and dot blot were performed to identify the ScFv in the supernatant and cell lysate. The gene of ScFv specific to hepatitis B virus core protein was sequenced. RESULTS: The ScFv screened from phage antibodies has a specific combination character with hepatitis B virus core antigen. Soluble ScFv was confirmed to express in E.coli. HB2151 and secrete in the supernatant. The sequence of ScFv gene conformed to that of heavy chain and kappa chain of human immunoglubulin. CONCLUSION: Human ScFv specific to hepatitis B virus core protein has been identified by means of the phage display technology, and its gene sequence has been determined.