Comparison between the suppression of tissue inhibitor of metalloproteinase-1 gene expression by recombinant adeno-associated virus carrying antisense RNA and small interfering RNA (siRNA) of TIMP-1 in rat hepatic stellate cells / 中华肝脏病杂志

<p><b>OBJECTIVES</b>Elevated tissue inhibitor of metalloproteinase-1 (TIMP-1) expression contributes to excess extracellular matrix in liver fibrosis. This study was designed to construct two recombinant adeno-associated viruses (AAV) carrying antisense RNA and small interfering RNA (siRNA) of TIMP-1 (rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo), and then to compare the suppression of TIMP-1 gene expression on rat hepatic stellate cell (HSC)-T6 cells infected by these two types of viruses in vitro.</p><p><b>METHODS</b>Antisense RNA amplified by rat HSC-T6 and U6 promoter followed by the annealing siRNA were cloned into the AAV vector (pdl6-95/neo) and packed in 293 cells to construct the recombinants rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo. Rat HSC-T6 cells were infected with these recombinant AAVs and selected by using G418, and real-time PCR after reverse transcription and Western blot were performed to detect the transcription and expression level of TIMP-1 gene in these cells.</p><p><b>RESULTS</b>The results of PCR, restrictive enzyme digestion and gene sequencing confirmed that the pdl6-95/ANTI-TIMP-1/neo and pdl6-95/siRNA-TIMP-1/neo had been reconstructed successfully. After they had been packed in 293 cells to form rAAV/ANTI-TIMP-1/neo and rAAV/siRNA-TIMP-1/neo, they were used to infect HSC-T6. Thirty days after the infection, the transcription level of TIMP-1 in HSC-T6 cells infected by rAAV/siRNA-TIMP-1/neo decreased dramatically compared with the mock control and normal HSC-T6 cells (P less than 0.01), and the expression level of TIMP-1 gene in HSC-T6 cells decreased significantly (60%), while the transcription and expression level of TIMP-1 in HSC-T6 cells infected by rAAV/ANTI-TIMP-1/neo had no significant difference with mock control and normal HSC-T6 cells (P more than 0.05).</p><p><b>CONCLUSION</b>RNA interference can exert a suppression of TIMP-1 gene in rat HSC, and when this function combines with AAV infection, it can suppress the specific gene expression for a long time by chromosomal integration.</p>

Recombinant adeno-associated virus carrying hepatitis B virus C gene (rAAV-HBV-C) effectively pulses dendritic cells (DC) without affecting their maturity / 中华肝脏病杂志

<p><b>OBJECTIVE</b>Recombinant virus pulsated dendritic cells (DCs) may affect their survival, growth and maturity. This study is to test the infection efficiency of recombinant adeno-associated virus carrying hepatitis B core antigen (rAAV-HBV-c) to DCs and the growth and maturity of them.</p><p><b>METHODS</b>Peripheral blood mononuclear cells (PBMCs) were isolated from healthy blood donors. Adherent monocytes were pulsed by rAAV-HBV-c and 293 lysate as controls on the first day of isolation. DCs were cultivated in AIM-V media with 1000 u/ml granulocyte macrophage stimulating factor (GM-CSF), 1000 u/ml interleukin-4 (IL-4) and 50 ng/ml tumor necrosis factor-alpha (TNFalpha) separately in vitro. DCs were examined at different times and the expressions of several clusters of differentiations (HLADR, CD14, CD80, CD83, CD86) were studied using FACS after being cultured for 7 days. The transcription and expression of HBV-C gene were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and intracellular staining fluorescence activated cell sorter (FACS), respectively.</p><p><b>RESULTS</b>The rAAV-HBV-c infected and uninfected monocytes gradually matured and their morphology had no significant differences. The CDs expressed on the surfaces of the two groups of DCs were also similar (HLADR: 96.1% vs. 94.5%; CD86: 87.7% vs. 89.8%; CD83: 75.6% vs. 78%; CD80: 52% vs. 54.3%; CD14: 6.4% vs. 4.5%). HBV-C gene mRNA expression was measured using RT-PCR and 89.5% of the rAAV-HBV-c infected DCs showed their protein expression using FACS.</p><p><b>CONCLUSION</b>rAAV-HBV-c can effectively pulse DCs without affecting the growth and maturity of them.</p>

Binding and inhibition of adeno-associated virus Rep78 protein with hepatitis B virus C promoter / 中华肝脏病杂志

<p><b>OBJECTIVES</b>Adeno-associated virus (AAV) Rep78 is known for its inhibitory effects on replication of several viruses and oncogenes transformations. The study was to investigate the effect of Rep78 on hepatitis B virus C (HBV-C) gene and the mechanism of it.</p><p><b>METHODS</b>HBV-C promoter and HBV-C gene with its promoter were amplified by PCR and labeled with 32P-ATP. Electrophoretic mobility shift assay (EMSA) and in vitro transcription were utilized to detect the binding of MBP-Rep78 with HBV-C promoter and the transcription of HBV-C gene.</p><p><b>RESULTS</b>EMSA showed that by increasing the amount of Rep78 protein from 0.1 microg to 1.0 microg, the binding bands got stronger in a dose-dependent manner. In addition, Rep78 antibody was used to certify the specificity of this binding. The compound of Rep78, Rep78 antibody and HBV-C promoter were seen as super shift bands in EMSA. Meanwhile, HBV-C gene transcription was significantly inhibited by in vitro transcription which meant that Rep78 could not only bind with HBV-C promoter, but also could inhibit the transcription of HBV-C gene.</p><p><b>CONCLUSION</b>AAV Rep78 could inhibit the transcription of HBV-C gene through its binding with HBV-C promoter.</p>

Sodium butyrate induces rat hepatic oval cells differentiating into mature hepatocytes in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To elucidate the effects of sodium butyrate on rat hepatic oval cell differentiation in vitro.</p><p><b>METHODS</b>Hepatic oval cells were isolated from rats fed with a choline-deficient diet supplemented with 0.1% (w/w) ethonine for 4 to 6 weeks. The cultured hepatic oval cells were identified by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). After hepatic oval cells were treated with sodium butyrate, the morphological changes were studied through Giemsa staining and the albumin expression level was tested by Western blot.</p><p><b>RESULTS</b>Immunohistochemical results showed the isolated cells were positive for both mature hepatocyte marker albumin and bile duct cell marker cytokeratin-19. Furthermore, RT-PCR results showed that the cells expressed stem cell marker c-kit, but not hematopoietic stem cell marker CD34. In short, the isolated cells were rat hepatic oval cells. 0.75 mmol/L sodium butyrate induced obvious phenotype changes of hepatic oval cells, including enlargement of the oval cells, a decrease in nucleus to cytoplasm ratio, and a 50% increase in the number of binucleated cells. Western blot results showed that 0.75 mmol/L sodium butyrate markedly raised the expression of albumin.</p><p><b>CONCLUSION</b>Sodium butyrate, a differentiation promoting agent, can induce rat hepatic oval cells (liver progenitor cells) to differentiate into mature hepatocytes in vitro.</p>