Study of proliferation ability of tumor antigen-loaded DC-CIK cells and its killing effect on hepatocarcinoma cells HepG2

Abstract

Objective To observe the proliferation ability of cocultured dendritic cells (DCs) loaded with tumor antigen and cytokine-induced killer cells (CIKs) and its killing effect on hepatocarcinoma cells HepG2. Methods The antigen of hepatocarcinoma cells HepG2 was prepared by repeated freezing and thawing of liquid nitrogen. Peripheral blood mononuclear cells (PBMNCs) were isolated from healthy donors by blood cell separator, then DCs and CIKs were induced. Ag-DCs were obtained by impinging DCs with tumor antigens. CIKs were divided into three groups: the first group was CIKs alone, the second group was mixed in the proportion of DCs∶CIKs=1∶5, and the third group was mixed in the proportion of Ag-DCs∶CIKs=1∶5. The three groups of cells were recorded as CIK group, DC-CIK group and Ag-DC-CIK group. The proliferation and cell phenotype of the three groups of cells were observed and the killing effects on hepatocarcinoma cells HepG2 were detected by methyl thiazolyl tetrazolium (MTT) assay. Results The proliferation multiples of the three groups of cells were gradually increased with the prolongation of culture time, and the proliferation rates of Ag-DC-CIK on the 9th day (61.32±1.72), the 12th day (190.83±3.53) and the 15th day (399.09±5.60) were significantly higher than those of CIK group (22.47±2.07, 55.91±1.81, 83.20±2.34) and DC-CIK group (40.26±2.49, 125.03±4.16, 251.55±3.25), and the difference between the three group was statistically significant (F=185.78, P=0.033; F=297.35, P=0.018; F=455.37, P<0.001), in addition, the differences between each two groups were statistically significant (all P<0.05). The cytotoxicity of Ag-DC-CIK to HepG2 cells at the effective target ratios of 5∶1 (31.71%±0.29%), 10∶1 (42.43%±1.86%) and 20∶1 (57.69%±1.11%) were significantly higher than those of CIK group (12.11%±1.14%, 21.30%±0.52%, 30.71%±1.26%) and DC-CIK group (20.06%±0.67%, 29.89%±1.37%, 39.11%±0.92%), and the difference between the three group was statistically significant (F=159.64, P=0.037; F=199.36, P=0.025; F=302.08, P<0.001), in addition, the differences between each two groups were statistically significant (all P<0.05). On the 15th day of cell culture, the flow cytometry analysis showed that all the three groups were expressed CD3+ CD8+ , CD3+ CD56+ double positive cells, the contents of CD3+ CD8+ 、CD3+ CD56+ double positive cells in the Ag-DC-CIK group (88.12%±1.24%, 61.35%±2.63%) were significantly higher than those in the CIK group (54.37%±3.08%, 18.22%±1.83%) and DC-CIK group (69.80%±1.46%, 39.51%±2.17%), and the difference between the three group was statistically significant (F=414.32, P<0.001; F=378.60, P<0.001), in addition, the differences between each two groups were statistically significant (all P<0.001). Conclusion The proliferation ability and killing effect of Ag-DC-CIK that obtained from antigen-pulsed DCs co-cultured with CIKs are significantly higher than those of CIKs and DC-CIKs. Key words: Dendritic cells; Killer cells; Cell line, tumor