Cytotoxic Activity Induced By Dendritic Cell Vaccine Plus Gemcitabine To The Hepatocellular Carcinoma Cancer Stem Cell

ObjectivePrimary liver cancer is the most common malignant tumor of the liver, as well as one of the most common malignant tumor all over the world. Primary liver cancer is a serious threat to human health. The etiology and pathogenesis was not entirely clear yet. The main risk factors are known to have hepatitis B virus and hepatitis C virus infection, aflatoxinB1ingestion, alcohol addiction, non-alcoholic liver disease and hereditary diseases. People with hepatitis B virus (HBV) infection are a major population at risk of developing HCC in China. Currently, there are93million Chinese who are HBV carriers, and about20million of them have chronic HBV infection. In recent years, researchers have found that cancer may be a kind of stem cell disease, cancer stem cells exist in a variety of malignancies, liver cancer is one of them. Cancer stem cells plays an important role in tumor initiating, tumor cell self-renewal and tumor metastasis. For the treatment of liver cancer, the main reason why we feel frustrated by the therapeutic effect is recurrence. Liver cancer recurrence after surgery may be due to the residual cancer stem cells. They may re-initiate tumor growth under certain conditions. Thus, therapy that target to the cancer stem cells may prevent the recurrence of hepatocellular carcinoma. With the development of molecular biology and immunology techniques, more and more attention is paid to the biological treatment of liver cancer. Immunotherapy mediated by dendritic cells has becoming a hot research topic. And there have been some positive development. CD133is an important cellular molecular markers of liver cancer stem cells. Our experiments were to sort CD133positive cells from Huh-7cell line, and then investigated the cytotoxicity of gemcitabine(GEM) combined with immunotherapy mediated by dendritic cells(DCs) loaded with CD133+liver cancer cell antigens to hepatocellular carcinoma cancer stem cells(HCC CSCs) in vitro.MethodsHuh-7cell line was choosed. The HCC CSCs were sorted from Huh-7cells in logarithmic growth phase by flow cytometery via the CD133molecular mark. Venous blood was from healthy adult. Blood cells were separated Lymphocyte separation medium. Peripheral blood mononuclear cells (PBMCs) were induced into dendritic cells in vitro. Then the DCs were sensitized by Huh-7and CD133+cancer cell antigens. Surface molecules expression was detected by flow cytometry before and after the DCs load antigen. Specific cytotoxic T lymphocytes were obtained after antigen loading-DCs were co-cultured with T cells. CD133+cells were taken as target cells for cytotoxicity tests. HCC CSCs were divided into five groups according to treatment factors:GEM group, CD133+-CTL group, GEM+CD133+-CTL group, Huh7-CTL group and GEM+Huh7-CTL group. The cytotoxic effects were assessed by CCK-8assay. Then variances were compared and analyzed.ResultsHuh-7cell growth was in good condition. CD133+cells were sorted out by flow cytometry, and such cells accounted for (3.0±0.78)%of the cell population. The separation efficiency was90%-99%.The DCs were adherent growth on the1st day, and the cell body was relatively small. The DCs were adherent loosely on the3ed day. The DCs showed a aggregation growth trend on day5. Most of the cells were in the growth situation of suspension or semi-suspension on day8.The GEM+CD133+-CTL group had the strongest cytotoxic effect to target cells, when compared with other groups. And the differences were statistically significant(P <0.05). In terms of the cytotoxic effect induced by the DC vaccine alone, the CD133+-CTL group was better than Huh7-CTL group(P<0.05).ConclusionThis study demonstrated the presence of CD133+liver cancer stem cells in Huh-7cell line, which accounts for only a small portion of the cell population. GEM combined with immunotherapy mediated by DCs loaded with CD133+liver cancer cell antigen could kill the HCC CSCs effectively. So this therapy may reduce the metastasis and recurrence after hepatectomy and liver transplantation and prompt a new strategy for the treatment of hepatocellular carcinoma.