Study On Cytotoxic Effect Of CIK Cells Activated By Alpha-Galactosylceramide Pulsed Dendritic Cells Against Hepatoma Carcinoma Cells

BackgroundDendritic cells is the most powerful antigen presenting cells in the body, play a unique status in the immune response induced. CIK cells is a new type immunocompetent cell, which is produced by a variety of cytokine induced killer cells. CIK cells have the anti-tumor activity of T cells and MHC restrictive kill tumor. CIK cells and DC cells are the application methods of adoptive immunotherapy. a-GalCer as an anticancer agent, antitumor is effective. Experiments show that, Relative to the separate injection a-GalCer, a-GalCer pulsed DC cells present a more antitumor activity. The activation of CIK cells by coculture with a-GalCer pulsed DC cells have not been studied, in this experiment, we study the proliferation activity and cytotoxic effect of CIK cells by coculture with a-GalCer pulsed DC cells.Objective To investigate the change of DC cell function and maturation pulsed by a-GalCer. To investigate the activate of NKT cells in peripheral blood by a-GalCer pulsed DC cells. To study the phenotype, proliferation activity and cytotoxic effect of CIK cells by coculture with a-GalCer pulsed DC cells.Methods 1.DC and CIK cells were generated from human peripheral blood mononuclear cells by density gradient centrifugation. Through the light microscope to observe the morphology and biological characteristics. Using flow cytometry to detect their immunophenotyp.2.Cell surface markers of DC pulsed by a-GalCer were assayed by flow cytometry and expression levels of DC function genes were detected by qRT-PCR.3.NKT cells were induced culture by DC and a-GalCer, using flow cytometry to detect the immunophenotyp and expression of cytokines levels.4.CIK cells were cocultured with a-GalCer pulsed DC cells, using flow cytometry to detect their cell phenotypes and trypan blue staining to detect CIK proliferation multiples. qRT-PCR was used to measure CIK function genes, meanwhile, CCK-8 kit was used to test cytotoxic effect of CIK cells.Results 1.CIK cells and mature DC cells were generated from human peripheral blood mononuclear cells. CD3+CD8+and CD3+CD56+overexpression in CIK cells, CD86, CD80, CD83 and CD11c overexpression in mature DC cells.2.a-GalCer could increase the expression of CD80, CD86, CD83, CDllc and also mRNA levels of CCR-7, IL-12, IL-10 for DC cells.3.NKT cells were induced culture by DC and a-GalCer, TCRVa24 TCRVp11 was overexpression in double positive NKT cells,secreting high level of IL-4 and INF-γ。4.CD3+CD56+expression level and proliferation activity of CIK cells were significantly increased by cocultured with a-GalCer pulsed DC cells.The mRNA levels of INF-y, IL-12, perform and particle enzyme B for CIK cells were increased too. The cytotoxicity rate in a-GalCer-DC-CIK group was highest, which was significantly higher than control group (P<0.01). The cytotoxicity rate of HepG2 cells in a-GalCer-DC-CIK group was highest, and the inhibitory rate was (59.17±4.35)%, which was significantly higher than control group (p<0.05).Conclusion a-GalCer pulsed could promote DC cells maturation. DC and a-GalCer induced obtain NKT cells in peripheral blood, activing NKT cells in vitro. CIK cells proliferation activity, maturation and cytotoxicity could highly enhance by cocultured with a-GalCer pulsed DC cells.