Cytotoxicity Effect Of DC Derived From Stem Cells Co-culture With CIK On K562/A02and Their Stem Cells

At present, we believe that the root cause of leukemia drug resistance and relapse is due to the remnants of the patient with leukemia stem cells. Traditional chemical drugs are generally only to remove most of the leukemia cells, but leukemia stem cells that has a self-protection mechanismare are not sensitive. Many chemotherapy drugs that we currently use can not radically cure leukemia. So we studied the treatment measures of the leukemia stem cell that is essential. In recent years, the biological immune therapy has become a hot topic. The immunotherapy research of DC and CIK against various tumor cell is very much. Many scholars believe that it has great potential for the treatment. The unique feature of this experiment is that leukemic stem cell-derived DC and CIK have the killing effect of the stem cells. It is to investigate the killing mechanism and provide some experimental basis for the study of treatment methods for LSC.ObjectivesThis study is to confirm that chronic myeloid leukemia (CML)-derived stem cells can generate dendritic cells (DC) and DC has the characteristics of CML stem cell. Analysis the mature immunophenotypes’changes of DC and CIK before and after the co-culture of DC and CIK, and to investigate the killing effect and proapoptosis effect of stem cell-derived Dendritic Cell (DC) co-cultured with cytokine induced killer (CIK) on K562/A02and its leukemia stem cell (LSC). This is to provide a theoretical basis and experimental basis for the removal of residual leukemia cells.MethodsBone marrow cells were isolated from the positive expression of BCR/ABL CML patients, and mononuclear cells were separated by Ficoll lymphocyte separation. Leukemic stem cells on CD34+CD38+were sorted and purified by flow sorting technology. DCs were amplified and induced by cytokines in vitro. The mononuclear cells from the same patient were induced to CIKs. DC and CIK were co-cultured acording to the ratio of1:10to determined proapoptosis and killing effect on leukemia stem cells of K562/A02cells by flow cytometry. Morphologies of cultured DC and CIK were examined under optical microscopy. Observe the ultrastructure of DC in the electron microscope. The expressions of DC’s and CIK’s markers typical、 P-gp resistance protein of LSC and DC were detected by flow cytometry. The expressions of BCR/ABL fusion gene of LSC and DC were detected by fluorescence in situ hybridization (FISH).Results1. Leukemia stem cells can be successfully induced to mature DC, and DC has the characteristics of CML stem cell.DC derived from LSC can be seen a large growth of dendritic burr, and it is typical dendritic cell morphology. They have the typical characteristics of mature DC by electron microscopy. The possitive expression rate of DC’s P-gp was about51.8%. BCR/ABL fusion gene positive rate is (48.87±18.44)%. Therefore DC has the characteristic of CML-LSC.2. The change of DC’s immunophenotypes after co-cultured with CIK.The expression rates of CD40, CD80, CD83, CD86and HLA-DR were increased in DC co-cultured with CIK. The expression rates of CD40, CD80, CD83and HLA-DR were increased from (41.08±2.35)%、(41.21±2.56)%、(32.28±3.19)%、(50.74±1.72)%、(49.65±2.29)%to(71.24±2.20)%、(62.42±3.03)%、(68.09±2.83)%、(71.28±2.66)%、(61.11±3.32)%. The differences were significant (P<0.01).3. The changes of CIK’s immunophenotypes after co-cultured with DC.The expression rates of CD3CD8and CD3CD56were increased in CIK co-cultured with DC. The mature immunophenotypes of CD3CD8and CD3CD56were increased from (46.65±3.91)%、(21.58±2.88)%to (59.35±3.77)%、(37.50±4.46)%. The differences were significant (P<0.01).4. Co-culture of DC and CIK can enhance the CIK’s killing effect on CD34+CD38+cells.After CIK group and DC-CIK group separately co-cultured with K562/A02cells, the proportion of CD34+CD38_cells is (3.30±0.27)%and (1.75±0.31)%by flow cytometry. The proportion of CD34+CD38-cells about the blank control group is (4.23±0.37)%. The differences were significant (F=64.470,P<0.01). The CIK group and DC-CIK group were lower than the blank control group, and DC-CIK group was lower the CIK group (P<0.01).5. Apoptosis-inducing effect of CIK and DC-CIK on K562/A02cells and their stem cells.CIK group and DC-CIK group induced apoptosis of K562/A02cells. Compared with CIKs, the apoptotic rate increased from (32.56±3.20)%to (40.21±3.09)%. The differences were significant (P<0.01). But it showed no evident effect on apoptosis of CD34+CD38-cells.ConclusionDC derived from stem cell has the characteristics of CML stem cell. DC co-cultured with CIK can effectively kill stem cells from multidrug resistant cells. they can induce apoptosis of K562/A02cells, but have no evident proapoptosis effect on stem cells.