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《Journal of Applied Clinical Pediatrics》 2009-15
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Biological Characteristics and Mechanism of Killing Tumor of Cytokine-Induced Killer Cells

LIU Miao,WU Xiao-yan,JIN Run-ming(Department of Pediatrics,the Union Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology,Wuhan 430022,Hubei Province,China)  
Objective To investigate the cytotoxicity and mechanism of killing tumor of cytokine-induced killer (CIK) cells in vitro.Methods Mononuclear cells were acquired freshly from bone marrow of children with leukemia,and the cells obstained were induced into dendritic cells by adding granulocyte-macrophage colony-stimulating,IL-4,TNF-α and other cytokines.Lymphocytes cells were isolated freshly from peripheral blood of children with leukemia by Ficoll-Hypaque density centrifugation,and the cells obstained were induced by IFN-γ,IL-2 and CD3McAb.The DC cells and CIK cells were co-cultured for 10-25 days,then DC-CIK cells were obtained.Phenotypes of DC-CIK were analyzed by flow cytomery.The cytotoxicity of DC-CIK against a variety of leukemic cell lines was investigated by MTT technique.When treated with mouse-anti-human LFA-1 monoclonal antibody,the expression of GATA-3 and T -bet in the levels of mRNA and protein were mea-sured by using RT-PCR and Western Blot technique,respectively.Results In the first 0-6 days,DC-CIK induced slowly,the proliferation of DC-CIK got 100-fold at the 13th day,cells were rapidly proliferating in the first 13-21 days.The maximum proliferation of DC-CIK reached at the 22nd day.The phenotypes of CD3,CD11a,CD54,HLA-DR were expressed highly; CD3/CD56,CD25,CD28,CD69,FasL were expressed moderately on DC-CIK.The expression of CD16 was not increased.DC-CIK possessed the cytotoxicity against tumor cells of B95,Jhhan and M07e.The effect was stronger to B95,there was no significant difference when the efficiency target ratio was 12.5:1.0 or 25:1,the cytotoxicity reached about 50% and 60%,respectively,against tumor cells of B95.However,it was not obvious to Jhhan and M07e.When the efficiency target ratio was 12.5:1.0 or 25:1,the cytotoxicity reached to 27.21%,25.13%,33.05%,29.72%,respectively,against tumor cells of Jhhan and M07e.When treated with mouse-anti-human LFA-1 monoclonal antibody,the expression of GATA-3 in the level of mRNA was up-regulated(t=3.425,4.523 Pa0.05),and the protein expression of GATA-3 was also increased(t=3.766,5.654 Pa0.05);and the expression of T-bet in the level of mRNA was down-regulated(t=4.682,3.898 Pa0.05),and the protein expression of T-bet was also decreased(t=5.624,4.982 Pa0.05).Conclusions IFN-γ,CD3McAb,IL-2 can induce peripheral lymphocytes to produce DC-CIK which exert highly efficient proliferation and cytotoxic effects on tumor cells.At the same time,it shows out the way that GATA-3 and T-bet were involved in the mechanism of killing tumor of DC-CIK cells induced by LFA-1/ ICAM-1.
【CateGory Index】: R73-3
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