Effects Of DC On The Biological Activity Of CIK Cells And Leukemia

Objective:The goal of the thesis is to find new approaches to treat acute leukemia with cellular immunotherapy. Co-culture dendritic cells (dendritic cells, DC) and cytokine activated killer cells (cytokine induced killer cells, CIK) and then observe the proliferation activity of the co-cultured DC-CIK cells, the changes in the immune cell membrane samples, the cytokines secretion and the effect of treating acute leukemia cells. At last, better immune active cells will be obtained through comparing the biological activity and the anti-leukemia effects of CIK with the features of the co-cultured DC-CIK cells. Methods:Draw healthy people's peripheral blood and culture CIK cells in vitro. After taking heparin to avoid blood clotting, use H-F (Hypaque-Ficoll) lymphocyte separation medium (relative density of 1.077±0.001) to isolate mononuclear cells (MNC), and then wash them with RPMI 1640 medium three times, adjusting the cell concentration to 4×106/ml. Collect the suspension cells in adherent culture and adjust the cell concentration to 1×106/ml,with 10% fetal calf serum (FBS) RPMI 1640 medium. Cultivate the cells with5% CO2 at 37℃, and then add different cytokines on day 0 and day 1.Thereafter change the culture medium every three days with rhIL-2 added. The way to cultivate Dendritic cells in vitro get healthy human peripheral blood mononuclear cells (MNC), then adjust cell concentration to 4×106/ml with RPMI 1640 medium. After 1-2 hours of nurturing, aspirate the medium and suspended cells. After putting fresh RPMI 1640 medium into the culturing flask, repeatedly blow and aspirte the cells adherent to the flask until they float. The cells will be collected and washed centrifugally, with the cells counted and the concentration regulated to 1×106/ml. After that, RPMI 1640 with 10% calf serum, rhGM-CSF550 U/ml, rhIL and 4500U/ml will be added. In accordance with the routine culture, medium is changed every half day, and cytokines is added. In the first 72 hours, add rhTNF-a 50U/ml to induce DC to maturation. On the 9th day, count DC and CIK cells by adopting trypan blue exclusion and adjust cell concentration of DC CIK and to 1×106/m and 5×106/ml respectively, with RPMI 1640 medium containing 10% calf serum. Have DC and CIKmixed,with the ratio of DC to CIK 1:5. Later, collect the mixture on the 3rd and the 6th day to exam the biological activity. Results:The results show:the co-cultured DC and CIK cells are obviously different from the simple CIK cells in the cell proliferation, cytotoxic activity, phenotype expression, cytokine secretion. Conclusion:The proliferation rate of CIK cells can increase.by being co-cultured with DC which can enhance CIK cells'proliferation activity and their anti-leukemia effects. There are more double-positive cells CD3+ CD8+ and CD3+ CD56+ in the co-cultured CIK cells. DC-CIK cells enjoy a higher level in cytokine secretion than the simple CIK cells.