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ObjectiveTo induce monocyte-derived dendritic cells(MoDC)from acute myeloid leukemia (AML) and healthy persons by rhCD40L in normal human AB serum system in vitro and to identify the morphology and phenotype of MoDC. MethodsPeripheral blood mononuclear cells(PBMNC)of AML and healthy persons were cultured in RPMI 1640 media including human AB serum, GM-CSF, rhIL-4 and rhCD40L, respectively. MoDC were identified by morphological features, surface antigen expression and the ability to stimulate T cells. ResultsAfter cultured for 7 days, MoDC displayed typical morphology with elongated dendritic process,and upregulation of the costimulatory molecules CD40,CD80,CD86 and CD83.The morphology and expression of costimulatory molecules were not significantly different between AML and healthy persons (P＞0.05),but were significantly different between rhCD40L group and without rhCD40L group (P＜0.05). MoDC had the ability to activate T cells, and there were no statistical differences between AML and healthy persons(P ＞0.05), but were significant differences between rhCD40L group and without rhCD40L group (P＜0.05). MoDC started to secrete IL-12 on day 5, and there was no statistical differences between AML and healthy persons(P＞0.05),and had differences between rhCD40L group and without rhCD40L group (P＜0.05).ConclusionMoDC can be cultured from the peripheral blood of AML and healthy persons.There were no significant differences in morphology and phenotype.Monocyted-derived DC can be used as an alternative to generate leukemia-specific cytotoxic T cells,especially in the presence of rhCD40L.
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Objective To explore the activity of thioredoxin reductase (TrxR) in chronic myeloid leukemia cell line K562 and the anti-leukemia effect of BBSKE (a novel inhibitor of TrxR) in vitro. Methods The activity of TrxR on K562 cell lineage and fresh bone marrow cell from healthy adult was analyzed by insulin reduction assay. The inhibition of proliferation was measured by CCK-8 assay. The anti-leukemia effect of BBSKE was detected by laser scanning confocal microscope,agarose gel electrophoresis and flow cytometry with Annexin V -FITC/PI staining. Results TrxR activity of K562 cell lineage was significantly higher than that of normal bone marrow mononuclear cells. The apoptosis of K562 cells could be induced at concentrations of 10 μmol/L BBSKE after treated for 24 hours. The typical DNA ladder bans were observed by agarose gel electrophoresis. The apoptotic rates of K562 cells were (10.28±2.74) %. Application of 10 μmol/L BBSKE for 48 hours could also induce apoptosis of fresh bone marrow cell from chronic myeloid leukemia patients, and the apoptotic rates were (5.70±0.48) %. Conclusion TrxR activity in chronic myeloid leukemia cells was significantly higher than that of normal cells. BBSKE inhibits the TrxR activity and the proliferation of K562 by inducing apoptosis.It might be a potential medication for chronic myeloid leukemia.
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Objective To establish a method to induce dendritic cells (DC) from peripheral blood mononuclear cells of healthy people in normal human AB serum in vitro and to identify the phenotype and the function of DC. Methods Peripheral blood mononuclear cells (PBMNC) of healthy people were cultured in RPMI 1640 media including human AB serum, GM-CSF, rhIL-4, and/no rhCD40 for 7 days to generate DC,which were identified by morphological features, surface antigen expression and the ability to stimulate T cells.Results After cultured and induced, DC displayed typical morphology with elongated dendritic process viewed by inverse light microscope as well as Wright-Gimsa stain. Mature DC express CD83 and the costimulatory molecules CD40 CD80, and CD86 to effectively activate T cells. In the five time points of 0 day, 1st day, 3rd day, 5th day and 7th day, the expression of CD83, CD40, CD80, CD86 and CD14 were significantly different (F= 50.253, 243.769, 248.181, 191.267 and 226.339, respectively, P＜ 0.05). The ability to stimulate T cells in GM-CSF, rhIL-4, and rhCD40L group was also stronger than that in GM-CSF and rhIL-4 group. DC started to secrete IL-12 from 5th day, the values were (42.92±1.54) pg/ml and (136.18±5.27) pg/ml in group of plus CD40L and of non plus CD40L, respectively. The secretion of the two groups of IL-12 were (60.09±2.27) pg/ml and (322.30±30.60) pg/ml (t = -44.941, -22.611, bath P ＜ 0.05). There are significant differences between the two groups. Conclusion DCs can be cultured from the peripheral blood of healthy people in normal human AB and rhCD40L serum.
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Objective To explore the expression level of thioredoxin (Trx) in acute myeloid leukemia (AML) cells,and its association with clinical features and prognosis,as well as the effects of diamide,an inhibitor of Trx,in inducing AML cells apoptosis.Methods Expression of Trx on AML cells and fresh bone marrow cells from healthy adults were analyzed by Western-blotting. The inhibition of proliferation was measured by MTT assay.The anti-leukemia effect of diamide was observed by morphology and agarose gel electrophoresis.Results 75 ％ (15/20) of AML patients expressed Trx,while no expression was observed in control group.Higher expression level of Trx was associated with higher WBC counts (x2 =9.375,P ＜ 0.05),which suggested that overexpression was associated with leukemogenesis.The inhibition of diamide on AML cells showed time and dose dependent by MTT assay.The IC50 values of diamide at 24 h,48 h and 72 h were 98.26 mg/ml,47.53 mg/ml and 8.34 mg/ml,respectively.After AML cells were treated with diamide,the apoptotic body appeared by morphology,and the typical DNA “ladder” bands were confirmed by agarose gel electrophoresis.Conclusion Trx expression level in AML cells is significantly higher than that of control group.Diamide inhibites the proliferation of AML cells by inducing apoptosis,which might be a potential agent for AML.
ABSTRACT
Thioredoxin reductase(TrxR) is an important component of thiol regulating system and contains a reactive and solvent accessible selenocysteine residue which is located on a flexible C-terminal arm of the protein and can combine various substrates and inhibitors. Many tumor cells have elevated TrxR levels and TrxR has also been shown to play a major role in drug resistance. A great number of effective natural and synthetic TrxR inhibitors are now availably possessing antitumor potential through induction of oxidative stress, cell cycle arrest and apoptosis. Inhibitors of TrxR may thus contribute to a successfully single, combinatory or adjuvant cancer therapy.