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<p><b>OBJECTIVE</b>To investigate the role of p38MAPK and PI3K/AKT pathways in mitomycin (MMC)-induced apoptosis in the liver stem-like cell line WB-F344.</p><p><b>METHODS</b>WB-F344 cells were exposed to MMC and apoptosis was evaluated by flow cytometry and DNA fragmentation. Phospho-MAPK and phospho-PI3K/AKT were detected by western blotting.</p><p><b>RESULTS</b>MMC induced apoptosis in WB-F344 cells at 6h after addition of MMC; the maximum level of apoptosis was reached at 24h after MMC exposure. The apoptosis effects of MMC were concentration dependent and inhibited when the PI3K pathway was abolished by the specific inhibitor LY294002, but not inhibited when the p38MAPK pathway was abolished by inhibitor SB203508.</p><p><b>CONCLUSION</b>Apoptosis of WB-F344 cells can be induced by MMC.Although MMC can activate both the PI3K/AKT and p38MAPK pathways, the apoptosis effect of MMC occurs via a PI3K pathway and is not dependent on the p38MAPK pathway.</p>
Subject(s)
Animals , Rats , Apoptosis , Blotting, Western , Cell Line , Chromones , Flow Cytometry , Mitomycin , Morpholines , Phosphatidylinositol 3-Kinases , Proto-Oncogene Proteins c-akt , Signal Transduction , p38 Mitogen-Activated Protein KinasesABSTRACT
<p><b>BACKGROUND</b>To explore the possibility of using retrovirus vector to carry HBV vector, and to prove that replication defective HBV could be normally packaged.</p><p><b>METHODS</b>Two kinds of full length of mutant HBV gene, which express dominant negative mutants, were inserted into retrovirus vector. After recombinant retroviruses were harvested, they were used to infect Hep G2 and 2.2.15 cell line. Then the expression of HBV core antigen in the Hep G2 cell was examined by immune fluorescence, and the existence of recombinant HB virion in the culture medium was examined by PCR.</p><p><b>RESULTS</b>High titer of recombinant retroviruses were obtained in the culture medium of transfected PA317 cell line. Core antigen was detectable in the recombinant retrovirus infected Hep G2 cell. Recombinant HB virion was detectable in the culture medium of recombinant retrovirus infected 2.2.15 cell.</p><p><b>CONCLUSIONS</b>The results suggested that recombinant retrovirus could carry HBV vector and express HBV products. When structural protein is offered by wt-HBV, the recombinant retrovirus may function as HBV vector, therefore it could be used in anti?HBV gene therapy.</p>
Subject(s)
Humans , Genetic Therapy , Genetic Vectors , Hepatitis B Core Antigens , Hepatitis B virus , Genetics , Recombination, Genetic , Retroviridae , Genetics , Tumor Cells, Cultured , Virus ReplicationABSTRACT
patients with refractory ascites were treated 921 times using B type ascites concentrating device made in our institute.The mean time of reinfusion was(2 3?0 9)h and the average volume of ascites withdrawn from abdominal cacity was(6 820?2 315)ml.The weight was reduced(6 7?2 4)kg.The urine volume in 24 hours increased from (257 8?235 6)ml to (725 8?436 9)ml( P
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Objective To explore the effect of the C gene truncated HBV mutant on HBV replication. Methods The C gene truncated HBV vector pHBV-?C was constructed through the molecular clone in vitro,and then transfected transiently into HepG2 cell.The expression of S protein was assayed by ELISA and SDS-PAGE Western blot.After co-transfection with pHBV-?C and wild HBV genome adwR9 into HepG2 cell the DNA was detected quantitatively by real-tine fluorescence quantitative PCR in the culture medium and the cell. Results There was no significant difference in expression of S protein assay by ELISA and Western blot.The DNA of the cotransfected group with pHBV-?C and adwR9 was lower than that of control group in the culture medium and the cell. Conclusions(C gene) truncated HBV mutant can cause the reduction of HBV replication.
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Purpose: To investigate the antitumor efficacy of intra-tumoral administration of plasmid DNA expressing mIL-12 in murine H22 liver tumor models grafted subcutaneously. Methods: Plasmid encoding mIL12 was constructed and examined the expression of cytokine in the eukaryotic cell through enzyme-linked immunosorbent assay (ELISA). The proliferation assay of T lymphoblasts was performed for measuring the biological activity of expressed mIL12. After intra-tumoral administration of plasmid DNA, mean diameters of the tumor mass and survival time were measured in each murine models group. Lactic dehydrogenase ( LDH) assay was performed to examine whether or not treatment with different plasmid DNA could induce systemic cytolytic activity of lymphocytes against parental H22 cells. Histopathological analysis was operated after administration of plasmid DNA vectors in each murine model group. Results: Growth of liver tumor was significantly inhibited( F =4. 10, P =0. 03), and activity of CTL against H22 was enhanced in mIL12 gene therapy group as compared with the control group. In the focal treated with pDC511mIL12 plasmid DNA, inflammatory cell infiltration was more extensive and necrosis was more definite than control group at 1 month after DMA injection. Conclusions: Intra-tumoral administration of plasmid DNA encoding interleukin-12 could inhibit the growth of H22 liver tumor and induce the host antitumor immune response efficiently in the murine model.
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Objective To explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular expression of antisense RNA. Methods Two parts of HBV genome were reversedly recombined back into overlength HBV genome, which can produce HBV particle, to express antisense RNA complementary to S or S promoter region respectively. HepG 2.2.15 cell lines were transfected with these constructs and the empty vector pMEP4, then positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA method and intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR. Results The mean inhibitory rates of HBsAg were (2.74?3.83)%、(66.54?4.45)%(P
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Objective To evaluate the replication, encapsulation and expression of the recombinant HBV vector with the truncated C gene. Methods C gene truncated HBV vectors were constructed by technologies of molecular clone and PCR based site-directed mutagenesis in vitro. The expression of GFP was observed with the fluorescence microscope after transfection of the recombinant HBV vector plasmids into HepG2 cells by using the liposome method. The capability of HBV vector replication, encapsulation and progeny virus production were examined with semi nested-PCR, native agarose gel electrophoresis blot, quantitative southern blot analysis and the routine PCR assays. Results The HBV vectors with truncated C gene were constructed successfully. The gene was expressed effectively after transfection. The C gene truncated HBV vectors could be replicated and encapsulated in hepatocytes with the helper virus. The encapsulated efficiency were as 4～8 folders as the non C gene truncated HBV vectors by quantitative analysis. In addition, the mature HBV particles carrying the interesting gene of GFP could secrete out from hepatocytes by the help of adjunctive vector. Conclusions The truncation of C gene could improve the encapsulation efficiency of HBV vectors with no effect on the replication and expression of the intact HBV vectors.
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The activity of plasma prolidase(PLD)was determined in patients with various chronic liver diseases.It was found that the activity of plasma PLD was increased significantly in 45 patients with cirrhosis and 31 patients with chronic active hepatitis.The plasma level of PLD was not correlated with the serum level of ALP.Plasma PLD was negatively correlated with serum albumin and positively correlated with serum gammablobulin.These findings suggest that the determination of the activity of plasma prolidase may be helpful in the diagnosis of chronic liver diseases.
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The clnical significance of serum glutathione S-transferase (GST) activity determination was evaluated by parallel measurements of GST and GPT activity inthe seraof 192 patients with liver diseases and 721 patients without liver diseas es. TheGST activity was significantly increased in the former, but it hadn't anychanges in patients without liver diseases. The highest GST activity was found in patients with severe hepatitis. In contrast to survival, the GST activity was persistently increased in those patients died later,and there appeared a "GST/GPT dissociation phenomenon". Although GPT activity had been normal in the convalescent stage of patients with hepatitis, the GST level in most of them was still high and different degree of pathological changes were also present in liver biopsy. Our data suggested that GST activity measurements might be a more sensitive parameter of liver cell damage than usual GPT measurements, and it might be valuable in evaluation of prognosis of severe hepatitis, and differentiation of hepa-tocellular jaundice from obstructive and hemolytic jaundice.
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Objective To study the role of NF-?B pathway in HGF (hepatocyte growth factor)-mediated proliferation of WB F-344 cell. Methods WB F-344 cells were treated with HGF in different concentrations i.e. 0, 20, 40, 80 and 100ng/ml. After incubation for 24 hours, proliferation effect was analyzed with [3H] Thymidine. WB F-344 cells transfected with I?B? plasmid mutants by liposome transfection were used to block NF-?B activity, and then they were treated with 40ng/ml HGF for 0, 15, 30, 60 and 120min, respectively. The nucleoprotein was extracted to determine the NF-?B DNA-binding activity by EMSA. WB F-344 cells were also transiently transfected with BD Great EscA PeTM SEAP vector with liposome and then stimulated with HGF in different concentrations (0, 10, 20, 40ng/ml) for 8 hours to detect the NF-?B transcription activity. WB F-344 cells were pre-treated with NF-?B inhibitor BAY-11-7082 or ASA, and then with 40 ng/ml HGF for 24 hours to analyze the proliferation effect by [3H] Thymidine. Results HGF promoted the proliferation of WB F-344 cells and exhibited positive dose-effect tendency. HGF could enhance the NF-?B DNA-binding activity. NF-?B complexes appeared 15min after addition of HGF. The complexes reached the peak value 30-60min after addition of HGF and then decreased gradually. HGF could also enhance the NF-?B transcription activity, exhibiting positive dose-effect tendency. HGF-mediated proliferation was remarkably inhibited when NF-?B activity was blocked by BAY-11-7082 or ASA. In WB F-344 cells transfected with p-CMV-I?B?, the proliferative response to HGF was completely interrupted. Conclusions HGF may promote the proliferation of WB F-344 cells in a positive dose-effect tendency. The activation of NF-?B is necessary for HGF-mediated proliferation of WB F-344 cells.
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0.05). The median diameter of the nodules in survival group was smaller (4.3?2.2cm)than that in death group(7.3?2.7cm, P0.05). Ten of 14 survived patients remained clinical free of tumor. In conclusion, RFA could extend survival time for patients with unresectable PHC.