| Objective The study used identifing the self-renewing stem / progenitor cells of CD34 leukemic cells in the bone marrow of patients with acute myeloid leukemia(AML),and to observe the difference between immature and mature dendritic cells cultured in peripheral blood of patients with complete remission.Study the effect of Calreticulin on autologous dendritic cells loaded with apoptoticacute myeloid leukemia CD34 +stem/ progenitor cells and to explore the possibility of enhancing the immunogenicity of leukemia cells in vitro.Methods The study selected the newly treated acute myeloid leukemia(except M3),CD34 should be positive in the immunological classification of leukemia.After signing the informed consent form with the patient,the bone marrow blood was collected from 3-5ml.the bone marrow blood was collected by density gradient centrifugation.Peripheral blood mononuclear cells(PBMC)were isolated from human lymphocytes and CD34 cells were separated by magnetic beads.The purity of CD34 cells was detected by flow cytometry.If CD34 cells can form colonies and identify their ability of self-renewal of dry / progenitor,they can carry out follow-up experiments.If the colony can not be formed,it is impossible to enter the group.The peripheral blood(autologous)of the patients with AML in complete remission phase is extracted.CD14 monocytes were isolated and separated by magnetic beads.The concentration of separated monocytes was adjusted to 2 × 10 6 / m L with RPMI 1640 medium containing 10% FBS.The cells were placed in a 24 well culture plate with GM-CSF 1000 U / ml,IL-4500U / ml,and the amount of media changed and cytokines were added every 48 hours.On the 5th day,DC maturation was observed and induced by TNF-a 50 U / ml.The phenotypic differences of mature and immature CD11 c and HLA-DR were analyzed by flow cytometry.Apoptosis of CD34 stem/ progenitor leukemia cells was induced by proteasome inhibitor(bortezomi)and anthracycline antibiotics(normodaunorubicin)in vitro.The fluorescent dye cell tracker orangeticus was divided into three groups,which were divided into three groups.After incubating on ice for 30 minutes,the cells were collected and labeled with CD11c-FITC antibody.After incubated on ice for 30 minutes,the cells were incubated with DC by 1: 2 for 2 hours,then the cells were collected and labeled with CD11c-FITC antibody.CD11 c and CTO double positive cells were detected by flow cytometry and the phagocytosis rate was calculated.Results 1)CD34 cells separated by magnetic beads from bone marrow mononuclear cells of primary acute myeloid leukemia were detected to be 92.67±2.015% by flow cytometry.2)the purified CD34 leukemia cells could form colonies on the culture medium.3)In the process of differentiation of CD14 monocytes into dendritic cells induced by DC GM-CSF and IL-4 from peripheral blood of complete remission phase,the cells were transformed from suspension to adherent cells and clustered into clusters.The colony size was not uniform.During 5 days of TNF-a culture,the cells were induced to mature.Dendritic cells with irregular morphology and a large number of spiny protrusions,the volume is obviously larger than before,most of them are suspended;The expression of HLA-DR and CD11 c on mature DC was higher than that on immature surface(P < 0.05).4)There was no significant difference in the phagocytosis rate of total recombinant calmodulin group and blank group,and the difference of anti-calcium reticulin antibody group was statistically significant.Conclusions1)the stem / progenitor cell character of CD34 cells in newly treated acute myeloid leukemia.2)GM-CSF and IL-4 could induce the differentiation of CD14 monocytes into dendritic cells.The mature DC could induce DC maturation,and the expression of CD11 c and HLA-DR on the mature DC was higher than that on the immature surface.3)reticulum Calreticulin,could enhance apoptosis of CD34 stem / progenitor leukemia cells loaded with autologous dendritic cells. |
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