The Specific Killing Effects Of The Whole Antigen Loaded D-CIK Against Liver Cancer Cell

Objective We tried to investigate the optimization of antigen loading types and the differences of the anti-tumor effects of D-CIK from different sources,, and provide a new way for the optimization of immunotherapy of HCC.Methods Tumor cell culture and preparation of tumor antigen We cultured primary hepatic carcinoma cells with tissue enzyme digestion method in vitro and the whole antigen and allogeneic hepatocellular carcinoma cell line(BEL-7402)antigens were prepared by repeated freezing and thawing.Induction culture、antigen loading and phenotype analysis of DC cells We used density gradient centrifugation to separate PBMC with 50 ml peripheral blood from patients.After 2hours culture, adherent cells can be induced to mature DC cells by GM-CSF, IL-4 and TNF-α.In the sixth day,wo add the tumor antigens into DC cells in order to load DC cells.Eighth DC cells were collected to detect the cell phenotype.Induction culture of CIK cells, D-CIK cell culture and phenotype analysis The non adherent cells were induced into CIK by IFN-,IL-2 and CD3 Mb cells.By day 8induce D-CIK cells by mixed culture of the antigen loaded DC cells and CIK cells,observe the proliferation of CIK cells dynamically.Fifteenth day CIK cells were collected for flow cytometry.Cell killing test of CIK CIKs was divided into three groups: simple CIKs, allogeneic Ag-D-CIK, allogeneic Ag-D-CIK.We detect the killing effect of three CIK cells against Hepatic celluler cancer cells and the dose effect relationship of their antitumor activity with MTT method.The target specificity of CIK cells was observed by the killing activity of liver cancer and breast cancer.Result The morphology and phenotype analysis of DC cells DC cells appeared cell processes, showing irregular cell morphology by day 3.Day 5cells were spindle or star shaped.Day 7 after loading and TNF- αinduced the cells became mature, the cells showed typical dendritic cell morphology.The results of flow cytometry showed that the expression rate of CD83, HLA-DR and CD80 was higher than that of the non loaded group.(p<0.01)The morphology, proliferation rate and phenotype analysis of CIK The initial CIK cells were small and round, with fewer cytoplasm and fewer cells,The proliferation of CIK cells began to change significantly in the third day, the cell size increased, the cytoplasm was abundant, and the number of cells began to increase,By day 8the proliferation rate was the highest after the addition of antigen loaded DC cells,21 st days later, the cell growth rate was obviously slowed down.Compared with the simple CIK group, the D-CIK with allogeneic antigen group, the expression of CD3+CD8+,CD3+CD56+ was the highest in the D-CIK with Autologous antigen group. the difference was statistically significant.Cell killing experiment There was a significant difference in the killing activity of CIK cells between the three groups when the target ratio was 5:1,Antigen loading group was larger than that of CIK group, and the self antigen loading group was higher than that of the homologous antigen group.The cell killing activity of each group was gradually increased with the increase of the target ratio in the range of 20:1.When the target ratio of 20:1, the cytotoxicity of autologous antigen loaded D-CIK on liver cancer cell(56.24±0.56%) is greater than that on the breast cancer cell(31.18±0.54%).Conclusion The whole antigen loading DC cells can induce the maturation of DC cells, increase the immune phenotype expression of DC cell and enhance the ability of antigen presentation of DC cells.The D-CIK from co culture of antigen sensitized DCs and CIKs has a more powerful ability of add value and lethality CIK cells induced by antigen loading play an anti tumor role with specificity ability.