AIM: To evaluate the expression of apoptosis related gene Fas ligand (FasL) in human hepatocellular carcinoma (HCC) cells HepG2 and its significance in apoptosis. METHODS: Levels of soluble Fas ligand (sFasL) in a gro...AIM: To evaluate the expression of apoptosis related gene Fas ligand (FasL) in human hepatocellular carcinoma (HCC) cells HepG2 and its significance in apoptosis. METHODS: Levels of soluble Fas ligand (sFasL) in a group of patients with hepatitis B virus (HBV)-induced chronichepatitis, HBV-positive liver cirrhosis and HCC were evaluated. In a further study, the recombinant eukaryotic expression plasmid pcDNA3.1hisB-FasL was transfected into HCC cells HepG2 by lipofection, and then soluble FasL was examined in the supernatant of culture cells by EIA, FasL expression in HepG2 cells was detected by immuohistochemistry. After being stained by annexin V and propidium iodine, cells were passed through a flow cytometer and examined by a fluorescence microscope and a laser scanning microscope.RESULTS: The sFasL levels were significantly lower in patients with HCC when compared to the patients with hepatitis or liver cirrhosis. In comparison with untransfected cells, the soluble FasL could be detected in the supernatant of transfected cells. FasL was expressed on the membranes and cytoplasm of transfected cells. The apoptotic cell rate was 36.30% in transfected cells, and was 11.53% in untransfected cells. Moreover, the different stage of apoptotic cells could be distinguished by annexin V and propidium iodine staining.CONCLUSION: Fas ligand is an apoptotic pathway of HCC cells.展开更多
AIM： To study the changes of human telomerase reverse transcriptase （hTERT） mRNA expression in human hepatocarcinoma cell lines （HepG2） and cholangiocarcinoma cell lines （QBC939） after HBx gene transfection and...AIM： To study the changes of human telomerase reverse transcriptase （hTERT） mRNA expression in human hepatocarcinoma cell lines （HepG2） and cholangiocarcinoma cell lines （QBC939） after HBx gene transfection and to illustrate the significance of transcriptional regulation of hTERT gene by HBx gene in the carcinogenesis. METHODS： HepG2 and QBC939 cell lines were cultured and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein （EGFP） coding sequence using lipid-mediated gene transduction technique. Thirty-six hours after transfection, EGFP expression in cells was used as the indicator of successful transfection. Flow cytometry was performed to determine the transfection efficiency. Cells were harvested and total RNA was extracted using TRIzol reagent. The expression of hTERT mRNA in HepG2 and QBC939 cell lines was assayed by reverse transcriptionpolymerase chain reaction. The expression of HBx protein in both cell lines was detected by immunocytochemical staining and Western blotting. RESULTS： Flow cytometry showed that the transfection efficiency was 46.4% in HepG2 cells and 29.6% in QBC939 cells for both HBx gene expression vector and blank vector. The expression of hTERT mRNA was meaningfully increased in HepG2 and QBC939 cell lines when transfected with HBx gene expression vector compared to those transfected with OPTI-MEM medium and blank vector. Immunocytochemical staining and Western blotting revealed HBx protein expression in HepG2 and QBC939 cells only when transfected with HBx gene. CONCLUSION： HBx gene transfection can upregulate the transcriptional expression of hTERT mRNA. The transactivation of hTERT gene by HBx gene is a newfound mechanism for pathogenesis of hepatocarcinomas and cholangiocarcinomas after HBV infection. 2005 The WJG Press and Elsevier Inc. All rights reserved展开更多
Objective. To investigate the anti- tumor effects of human single chain interleukin- 12 (hscIL- 12). Method. pcDNA/ hscIL- 12 recombinant was transfected into human hepatic carcinoma cells (7721 cells) by lipofectin m...Objective. To investigate the anti- tumor effects of human single chain interleukin- 12 (hscIL- 12). Method. pcDNA/ hscIL- 12 recombinant was transfected into human hepatic carcinoma cells (7721 cells) by lipofectin method. The 7721/hscIL- 12 cells which secrete hscIL- 12 stably, were obtained via G418 selection, and in vitro the influence of hscIL- 12 gene transduction on the growth of tumor cells was evaluated by cell cycle analysis. In vivo, genetically engineered 7721 cells (7721/hscIL- 12, 7721/pcDNA) and parental cells were implanted into BALB/c nude mice,respectively. 7721/pcDNA and 7721/hscIL- 12 groups were divided into two sub- groups on day 8: one was administered with hPBL twice, 6 days at interval; the other was given equal volume of PBS. Mice were sacrificed on day 26, and spleens and tumors were taken out for histologic assay. Results. hscIL- 12 produced stably by 7721/hscIL- 12 cells had bioactivity, and it was proved by Western blot, immunocytochemistry, and in situ hybridization. In vitro, compared with 7721 and 7721/pcDNA, the 7721/hscIL- 12 grew much more slowly. FACS assay showed apparent G1 arrest of 7721/hscIL- 12 cells. In animal experiment, on day 8 after inoculation, the tumors of 7721 and 7721/pcDNA group were up to 5～ 7mm,while those of 7721/hscIL- 12 group were 2～ 4mm.When treated with hPBL, the tumor of 7721/hscIL- 12 group disappeared completely. Histologically, the tumors from 7721/hscIL- 12 without hPBL treatment had numerous lymphocyte infiltration, the tumor cells displayed depression looking, atrophy, focal necrosis and apoptosis , whereas the tumors of 7721 and 7721/pcDNA groups grew thrivingly. Conclusion. hscIL- 12 transduced 7721 cells could induced significant antitumor immune response which resulted in tumor regression totally when the hPBL was inoculated, and also hscIL- 12 has certain effects on mice immune system. These findings suggest that hscIL- 12 and hscIL- 12 gene therapy might have promising prospects in clinical application.展开更多
Objective To investigate the effect of overexpression of bcl-2 on ethanol-induced apoptosis of primary hepatocellular carcinoma (HCC) cells.Methods The retrovirus expression vector pDOR-SB containing human bcl-2 cDN...Objective To investigate the effect of overexpression of bcl-2 on ethanol-induced apoptosis of primary hepatocellular carcinoma (HCC) cells.Methods The retrovirus expression vector pDOR-SB containing human bcl-2 cDNA was introduced into a human HCC cell line HCC-9204 by liposome-mediated transfection. pDOR-transfected and non-transfected HCC-9204 cells were used as controls. The expression of Bcl-2 protein by transfected HCC-9204 cells was detected by the immunohistochemical method. Then the cells were cloned with the limited dilution method continually until a monoclonal cell strain whose positive rate of Bcl-2 protein was 100% detected by flow cytometry was obtained. The killing rates of cells were detected by Methabenzthiazuron assay after the treatment of 6% ethanol for 6?h. The extent of apoptosis was analyzed by transferase-mediated dUTP nick end labeling (TUNEL) staining and flow cytometry.Results Most of the pDOR-SB-transfected cells demonstrated Bcl-2 positive signals, while no signal was found in the controls. The positive rate of Bcl-2 protein detected by flow cytometry in the obtained monoclonal cell strain, which was named HCC-bcl2, was 100% after the cells had been cloned 3 times continually. The killing rate, TUNEL index and the scale of sub-G 1 apoptotic peak in HCC-bcl2 cells were all significantly lower than those in the control cells.Conclusion Overexpression of Bcl-2 protein suppresses ethanol-induced apoptosis of the HCC cell line HCC-9204.展开更多
基金Supported by the Foundation of Health Department of Hunan Province, No. 9683
文摘AIM: To evaluate the expression of apoptosis related gene Fas ligand (FasL) in human hepatocellular carcinoma (HCC) cells HepG2 and its significance in apoptosis. METHODS: Levels of soluble Fas ligand (sFasL) in a group of patients with hepatitis B virus (HBV)-induced chronichepatitis, HBV-positive liver cirrhosis and HCC were evaluated. In a further study, the recombinant eukaryotic expression plasmid pcDNA3.1hisB-FasL was transfected into HCC cells HepG2 by lipofection, and then soluble FasL was examined in the supernatant of culture cells by EIA, FasL expression in HepG2 cells was detected by immuohistochemistry. After being stained by annexin V and propidium iodine, cells were passed through a flow cytometer and examined by a fluorescence microscope and a laser scanning microscope.RESULTS: The sFasL levels were significantly lower in patients with HCC when compared to the patients with hepatitis or liver cirrhosis. In comparison with untransfected cells, the soluble FasL could be detected in the supernatant of transfected cells. FasL was expressed on the membranes and cytoplasm of transfected cells. The apoptotic cell rate was 36.30% in transfected cells, and was 11.53% in untransfected cells. Moreover, the different stage of apoptotic cells could be distinguished by annexin V and propidium iodine staining.CONCLUSION: Fas ligand is an apoptotic pathway of HCC cells.
文摘AIM： To study the changes of human telomerase reverse transcriptase （hTERT） mRNA expression in human hepatocarcinoma cell lines （HepG2） and cholangiocarcinoma cell lines （QBC939） after HBx gene transfection and to illustrate the significance of transcriptional regulation of hTERT gene by HBx gene in the carcinogenesis. METHODS： HepG2 and QBC939 cell lines were cultured and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein （EGFP） coding sequence using lipid-mediated gene transduction technique. Thirty-six hours after transfection, EGFP expression in cells was used as the indicator of successful transfection. Flow cytometry was performed to determine the transfection efficiency. Cells were harvested and total RNA was extracted using TRIzol reagent. The expression of hTERT mRNA in HepG2 and QBC939 cell lines was assayed by reverse transcriptionpolymerase chain reaction. The expression of HBx protein in both cell lines was detected by immunocytochemical staining and Western blotting. RESULTS： Flow cytometry showed that the transfection efficiency was 46.4% in HepG2 cells and 29.6% in QBC939 cells for both HBx gene expression vector and blank vector. The expression of hTERT mRNA was meaningfully increased in HepG2 and QBC939 cell lines when transfected with HBx gene expression vector compared to those transfected with OPTI-MEM medium and blank vector. Immunocytochemical staining and Western blotting revealed HBx protein expression in HepG2 and QBC939 cells only when transfected with HBx gene. CONCLUSION： HBx gene transfection can upregulate the transcriptional expression of hTERT mRNA. The transactivation of hTERT gene by HBx gene is a newfound mechanism for pathogenesis of hepatocarcinomas and cholangiocarcinomas after HBV infection. 2005 The WJG Press and Elsevier Inc. All rights reserved
文摘Objective. To investigate the anti- tumor effects of human single chain interleukin- 12 (hscIL- 12). Method. pcDNA/ hscIL- 12 recombinant was transfected into human hepatic carcinoma cells (7721 cells) by lipofectin method. The 7721/hscIL- 12 cells which secrete hscIL- 12 stably, were obtained via G418 selection, and in vitro the influence of hscIL- 12 gene transduction on the growth of tumor cells was evaluated by cell cycle analysis. In vivo, genetically engineered 7721 cells (7721/hscIL- 12, 7721/pcDNA) and parental cells were implanted into BALB/c nude mice,respectively. 7721/pcDNA and 7721/hscIL- 12 groups were divided into two sub- groups on day 8: one was administered with hPBL twice, 6 days at interval; the other was given equal volume of PBS. Mice were sacrificed on day 26, and spleens and tumors were taken out for histologic assay. Results. hscIL- 12 produced stably by 7721/hscIL- 12 cells had bioactivity, and it was proved by Western blot, immunocytochemistry, and in situ hybridization. In vitro, compared with 7721 and 7721/pcDNA, the 7721/hscIL- 12 grew much more slowly. FACS assay showed apparent G1 arrest of 7721/hscIL- 12 cells. In animal experiment, on day 8 after inoculation, the tumors of 7721 and 7721/pcDNA group were up to 5～ 7mm,while those of 7721/hscIL- 12 group were 2～ 4mm.When treated with hPBL, the tumor of 7721/hscIL- 12 group disappeared completely. Histologically, the tumors from 7721/hscIL- 12 without hPBL treatment had numerous lymphocyte infiltration, the tumor cells displayed depression looking, atrophy, focal necrosis and apoptosis , whereas the tumors of 7721 and 7721/pcDNA groups grew thrivingly. Conclusion. hscIL- 12 transduced 7721 cells could induced significant antitumor immune response which resulted in tumor regression totally when the hPBL was inoculated, and also hscIL- 12 has certain effects on mice immune system. These findings suggest that hscIL- 12 and hscIL- 12 gene therapy might have promising prospects in clinical application.
文摘Objective To investigate the effect of overexpression of bcl-2 on ethanol-induced apoptosis of primary hepatocellular carcinoma (HCC) cells.Methods The retrovirus expression vector pDOR-SB containing human bcl-2 cDNA was introduced into a human HCC cell line HCC-9204 by liposome-mediated transfection. pDOR-transfected and non-transfected HCC-9204 cells were used as controls. The expression of Bcl-2 protein by transfected HCC-9204 cells was detected by the immunohistochemical method. Then the cells were cloned with the limited dilution method continually until a monoclonal cell strain whose positive rate of Bcl-2 protein was 100% detected by flow cytometry was obtained. The killing rates of cells were detected by Methabenzthiazuron assay after the treatment of 6% ethanol for 6?h. The extent of apoptosis was analyzed by transferase-mediated dUTP nick end labeling (TUNEL) staining and flow cytometry.Results Most of the pDOR-SB-transfected cells demonstrated Bcl-2 positive signals, while no signal was found in the controls. The positive rate of Bcl-2 protein detected by flow cytometry in the obtained monoclonal cell strain, which was named HCC-bcl2, was 100% after the cells had been cloned 3 times continually. The killing rate, TUNEL index and the scale of sub-G 1 apoptotic peak in HCC-bcl2 cells were all significantly lower than those in the control cells.Conclusion Overexpression of Bcl-2 protein suppresses ethanol-induced apoptosis of the HCC cell line HCC-9204.
