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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 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.
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AIM: To study the effects of [Gly~(14)]-humanin,one of the strongest derivate of humanin,on proliferation and differentiation of neural stem cells(NSCs) and the protective action of cell death or apoptosis induced by ?-amyloid protein 1-42.METHODS: NSCs were treated with different concentrations of [Gly~(14)]-humanin and ?-amyloid protein 1-42.RESULTS: 10 nmol/L [Gly~(14)]-humanin made NSCs resistant to the apoptotic action induced by A?P1-42 and prevented NSCs from death induced by 25 ?mol/L ?-amyloid protein 1-42.The differentiated neural stem cells yield more neuronal cells than that in control groups when 10 nmol/L [Gly~(14)]-humanin was added to the culture media.The number of cells increased and the cultures grown with a manner of floating cell clones likes that cultured in the presence of mitogen when 100 nmol/L [Gly~(14)]-humanin was added to the differentiation culture media.CONCLUSION: The [Gly~(14)]-humanin significantly promoted the proliferation and neuronal differentiation of neural stem/progenitor cells and also inhibited the toxic action of ?-amyloid protein 1-42 on cultured neural stem/progenitor cells.
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AIM: To study the effects of ?-amyloid protein (A?) on neural stem cells cultured in vitro. METHODS: Neural stem cells (NSC) were isolated from E13 SD rats and cultured in serum-free medium (DMEM/F12). After detected by nestin, the A? was added to the NSC medium to observe the viability and proliferation of NSC by MTT, cell count and flow-cytometric examination. The effects of A? on differentiated NSC were also observed. RESULTS: A? markedly inhibited the proliferation and the cell viability of NSC when its concentration was higher than 25 ?mol/L. The differentiatory ability of NSC was inhibited when A? was in very low concentration. CONCLUSION: A? significantly inhibits the proliferation and differentiation of NSC and this may be one of the reasons that Alzheimer's disease is induced. [