ABSTRACT
OBJECTIVE@#To observe expression of SIRT3 in normal liver tissue, cirrhotic tissue and hepatocellular carcinoma (HCC) tissues, and to explore the significance of SIRT3 in primary HCC.@*METHODS@#SIRT3 expression was detected in 10 normal cases, 30 cases with, 30 HCC cases by immunohistochemical and Western-blotting method.@*RESULTS@#Immunohistochemical assay showed that the SIRT3 positive expression rates were 100.0% (10/10), 96.7% (29/30) and 60.0% (18/30), respectively in normal group, paracancer group and HCC group. And the SIRT3 expression in HCC group was significantly lower than in normal group and paracancer group (P<0.05). Western-blotting showed the SIRT3 expression in cancer tissue was 0.29±0.07, significantly lower than that in paracancer group and normal group (P<0.05). SIRT3 expression was related to the differentiation degree and portal vein tumor thrombus (P<0.05).@*CONCLUSIONS@#Abnormal expression of SIRT3 is closely related to the biological behavior of primary HCC.
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Carcinoma, Hepatocellular , Pathology , Cell Proliferation , Hepatocytes , Physiology , Immunohistochemistry , Liver , Pathology , Liver Cirrhosis , Pathology , Liver Neoplasms , Pathology , Neoplasm Invasiveness , Pathology , Sirtuin 3ABSTRACT
<p><b>OBJECTIVE</b>To investigate if granulocyte-macrophage colony stimulating factor (GM-CSF) gene-modified dendritic cells (DC) enhance antitumor immunity in vitro.</p><p><b>METHODS</b>Mice were injected with chemokine ligand 3 (CCL3) via the tail vein. Fresh B220(-)CD11c(+) cells were sorted from the peripheral blood mononuclear cells (PBMCs) and cultured into DCs by cytokines.DCs were transfected with AdGM-CSF gene at different ratios of multiplicity of infection (MOI) to determine the optimal gene transfection conditions, and the expression of GM-CSF was detected after transfection. The variation of GM-CSF gene-modifiedDCs were analyzed by morphological examination, phenotype analysis, and mixed lymphocyte reaction (MLR).DCs were loaded with gastric cancer antigen obtained by freezing and thawing method. The killing effect of DCs vaccine-stimulated T lymphocytes on gastric cancer cells was assessed by MTT assay. INF-gamma production was determined with the INF-gamma ELISA kit.</p><p><b>RESULTS</b>B220(-)CD11c(+) cells increased obviously after CCL3 injection. The ELISA results showed that after GM-CSF gene modification, DCs could produce high level of GM-CSF. When DCs were transfected with AdGM-CSF gene at MOI equal to 100, the GM-CSF level in culture supernatants reached saturation [(130.00 +/- 12.61) pg/ml]. After GM-CSF gene-modification, DCs tend to be more maturated as detected by morphological observation and phenotype analysis. At the same time, the capacity of activating the proliferation of allogeneic T lymphocytes was enhanced greatly. T lymphocytes stimulated by DCs transfected with GM-CSF gene showed a specific killing effect on gastric carcinoma cells and produced high level of INF-gamma [(1245.00 +/- 13.75) pg/ml].</p><p><b>CONCLUSION</b>After GM-CSF gene modification, DCs can produce high level of GM-CSF, which tend to be more maturated, and the capacity of activating the proliferation of allogeneic T lymphocytes is enhanced greatly. GM-CSF gene modified DCs can induce specific CTL to target tumor cells in vitro.</p>
Subject(s)
Animals , Female , Mice , Adenoviridae , Genetics , B7-1 Antigen , Metabolism , B7-2 Antigen , Metabolism , CD40 Antigens , Metabolism , Cancer Vaccines , Allergy and Immunology , Cell Line, Tumor , Cell Proliferation , Cytotoxicity, Immunologic , Dendritic Cells , Cell Biology , Allergy and Immunology , Metabolism , Granulocyte-Macrophage Colony-Stimulating Factor , Genetics , Metabolism , Histocompatibility Antigens Class II , Metabolism , Interferon-gamma , Bodily Secretions , Mice, Inbred BALB C , Recombinant Proteins , Stomach Neoplasms , Allergy and Immunology , Metabolism , Pathology , T-Lymphocytes, Cytotoxic , Cell Biology , Allergy and Immunology , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To investigate the anti-gastric carcinoma immunological efficacy of dendritic cells (DC) precursors, that were mobilized into the peripheral blood by injection of macrophage inflammation protein-1 alpha (MIP-1 alpha), and induced by DC vaccine expressing melanoma antigen gene-3 (MAGE-3) ex vivo and in vivo.</p><p><b>METHODS</b>615 mice were injected with MIP-1 alpha via the tail vein. Freshly isolated B220(-) CD11c+ cells were cultured with cytokines and assayed by phenotype analysis and mixed lymphocyte reaction (MLR). For adenoviral (Ad)-mediated gene transduction, cultured B220(-) CD11c+ cells were incubated with Ad-melanoma antigen gene-3. MIP-1 alpha-mobilized B220(-) CD11c+ cells pulsed MFC cells tumor lysate were used as positive control. The stimulated DC vaccination-induced T lymphocytes, and the killing effect of the T cells on gastric carcinoma cells were assayed by MTT. INF-gamma production was determined with the INF-gamma ELISA kit. To establish the solid tumor model, groups of 615 mice were injected with MFC cells subcutaneously into the abdominal wall. MIP-1 alpha-mobilized DC vaccines expressing MAGE-3 gene were used to immunize the mice after the challenge of MFC cells, then the tumor size and the survival of mice were examined to detect the therapeutic effect of DC vaccines.</p><p><b>RESULTS</b>B220(-) CD11c+ cells increased obviously after MIP-1 alpha injection, and freshly isolated B220(-) CD11c+ cells cultured with mGM-CSF, IL-4, and mTNF-alpha were phenotypically identical to typical DC, gained the capacity to stimulate allogeneic T cells. These MIP-1 alpha-mobilized DCs were transduced with Ad-MAGE-3, which were prepared for DC vaccines expressing tumor antigen. T lymphocytes stimulated with DC-transduced with Ad-MAGE-3 showed specific killing effect on gastric carcinoma cells and produced high levels of INF-gamma [(1460.00 +/- 16.82) pg/ml]. Five days after the MFC cells challenge, the mice were subsequently injected with DC vaccines. The tumor size of the experimental group was significantly smaller than that in the positive control group and the negative control groups (P<0.01). Kaplan-Meier survival curves showed the survival of the experimental group mice was significantly longer than that of the control groups (P<0.01).</p><p><b>CONCLUSION</b>B220(-) CD11c+ DC precursors are rapidly accumulated in the peripheral blood after injection of MIP-1 alpha into mice, which can further differentiate into mature DCs. These MIP-1 alpha-mobilized DCs, when transduced with MAGE-3 gene, can induce specific CTL to gastric carcinoma cells ex vivo, and can generate anti-tumor therapeutic effects on MFC cells loading mice in vivo. The efficiency of anti-tumor therapeutic immunity induced by MIP-1 alpha-mobilized DCs expressing tumor antigen are much more potent than MIP-1 alpha mobilized DCs pulsed MFC cells tumor lysate.</p>