Study On The Cytotoxicity Of CIK Activated By DCs Cultured In Serum-free Medium Against Hepatocellular Carcinoma In Vitro

Objectives:1.To establish a highly effective method for preparing the DCs vaccine in serum-free medium in vitro,and provid experimental proof for the clinical antitumor therapy.2.To explore the effect of DCs on the proliferation of CIK and the cytotoxicity of CIK against hepatocellular carcinoma cells.Methods:1.The mononuclear cells separated from cord blood were induced to differentiate into the immature DC by rhGM-CSF and rhIL-4 in serum-free medium (SFM)or 5%fetal calf serum RPMI-1640 medium(SM).TNF-αand the Freeze-thaw lysates of hepatocellular carcinoma cells were was added on the 5th day. On the 7th day,these cells were harvested and identified with light microscope and flow cytometry(FCM).Then,the morphology and surface markers of SM-cultured DCs(SM-DCs)and SFM-derived DCs(SFM-DCs)was compared.2.The mononuclear cells from cord blood were induced to differentiate into CIK by cytokines IFN-γ,IL-2,CD3-Ab,IL-la.DCs were co-cultured with autologous CIK cells on the 7th day.Then,the proliferation and the cytotoxicity of CIK stimulated with DCs was examined by methyl thiazoly tetrazolium(MTT).Results:1.Mononuclear cells separated from the cord blood could be induced into typical and phenotypic DC s with the compatibility of such cellular factors as rhGM-CSF, IL-4,TNF-α,and pulsed antigen.The flow cytometry detection showed:the expression level(%)of cell surface markers of DCs cultured on the SFM-DC:CD1a 56.6±3.8,CD11c 64.5±4.3,CD14 19.5±1.8,CD40 77.8±7.5,CD83 70.8±5.1,CD86 76.5±6.3 HLA-DR 91.5±3.5;The expression level(%)of cell surface markers on the SM-DC:CD1a 59.3±5.1,CD11c 59.6±6.2,CD14 20.1±2.2,CD40 75.6±6.4, CD83 65.2±4.4,CD86 80.4±6.9,HLA-DR 94.5±2.8;The levels of surface markers were not significantly different between SFM-DC and SM-DC,(p＞0.05).2.The results of mixed lymphocyte reaction:The average stimulator index(SI) of CIK actvavted by SFM-DC:2.01±0.22;The average stimulator index(SI)of CIK actvavted by SM-DC:2.31±0.24.The difference of the stimulator index(SI)between SFMDC and SMDC was not significant and they could effectively activate CIK cells to proliferate.3.The results of cytotoxicity assay:The cytotoxicity to hepatocellular carcinoma cells:the cytotoxicity of SFMDC-CIK 74.5±7.5,the cytotoxicity of SMDC-CIK 77.9±6.0,the cytotoxicity of SMDC-CIK 54.1±4.9;The statistical analysis shows:①The difference of the cytotoxicity between SFMDC-CIK and SMDC-CIK was not significantly,(p＞0.05);②SFMDC-CIK and SMDC-CIK possessed higher ability to lyse hepatoma cells than inactivated CIK,(p＜0.05).Conclusion:1.We Preliminarily established a method for preparing the DCs vaccine in serum-free medium in vitro.In serum-free DCs medium with rhGM-CSF 100ng/ml, rhIL-4 50ng/ml,TNF-α50ng/ml,mononuclear ceils from the cord blood could be induced into the Clinical grade DCs with typical modality and Cell surface markers2.Both SFM-DC and SM-DC could Effectively activate CIK cells to have higher proliferative and anti-tumor activity,and DC-CIK will have a good prospect in the treatment of tumors.