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BACKGROUND: Dendritic cell is the most major antigen presenting cell of organism. It is proved in recent studies that human umbilical cord blood mononuclear cells induced and cultured in vitro by recombinant human granulocyte-macrophage colony stimulating factor (rhG-MCSF) and recombinant human interleukin-4(rhIL-4) can generate a great many dendritic cells and promote the lethal effect of T cells on human neuroblastoma, but it is unclear that whether the lethal effect is associated with the most proper concentration of dendritic cells. OBJECTIVE: To investigate the lethal effect of human umbilical cord blood mononuclear cells induced in vitro by cytokines differentiating into dendritic cells on human neuroblastoma, and its best concentration range. DESIGN: Open experiment. SETTING: Department of Pediatrics, the Medical School Hospital of Qingdao University. MATERIALS: The study was carried out in the Shandong Provincial Key Laboratory (Laboratory for the Department of Pediatrics of the Medical School Hospital of Qingdao University) during September 2005 to May 2006. Human umbilical cord blood samples were taken from the healthy newborn infants of full-term normal delivery during October to November 2005 in the Medical School Hospital of Qingdao University, and were voluntarily donated by the puerperas. Main instruments: type 3111 CO2 incubator (Forma Scientific, USA), type 550 ELISA Reader (Bio-Rad, USA). Main reagents: neuroblastoma cell line SK-N-SH (Shanghai Institute of Life Science, Chinese Academy of Sciences), RPMI-1640 culture fluid and fetal bovine serum (Hyclone), rhIL-4 (Promega, USA), rhG-MCSF (Harbin Pharmaceutic Group Bioengineering Co.Ltd), rat anti-human CD1a monoclonal antibody and FITC-labeled rabbit anti-rat IgG (Xiehe Stem cell Gene Engineering Co.Ltd). METHODS: ① Human umbilical cord blood mononuclear cells obtained with attachment methods differentiated into human umbilical cord blood dendritic cells, presenting typical morphology of dendritic cells after in vitro induction by rhG-MCSF and rhIL-4. ② Different concentrations of dendritic cells[ dendritic cells: neuroblastoma cells=20∶1,50∶1,100∶1(2×108 L-1,5×108 L-1,1×109 L-1)], 1×109 L-1 T cells and 1×107 L-1 neuroblastoma cells were added in the experimental group. 1×109 L-1 T cells and 1×107 L-1 neuroblastoma cells were added in the control group. ③ Main surface marker CD1a molecules of dendritic cells were detected with indirect immunofluorescence, and the percent rate of dendritic cells was counted with ultraviolet light and expressed as the expression rate of CD1a+ cells. ④ Single effector cells and target cells were respectively set in the experimental group and control group to obtain the lethal effect. The lethal effect of dendritic cells on neuroblastoma cells was indirectly evaluated by detecting cellular survival with MTT assay. The lethal effect(%)=(1-A experimental well-A effector cell well/A target cell well)×100%.⑤The experimental data were presented as Mean ±SD, and paired t test was used. MAIN OUTCOME MEASURES: ① Morphological characters of dendritic cells in the process of induction and differentiation. ②CD1a+ cellular expression rate. ③Lethal effect of dendritic cells on neuroblastoma cells. RESULTS: ①Morphological characters of dendritic cells in the process of induction and differentiation: On the 15th day after human umbilical cord blood mononuclear cells were induced by rhG-MCSF and rhIL-4, typical morphology of dendritic cells could be seen under an inverted microscope. ②Expression rate of CD1a+ cells was (43.12±5.83)%. ③Lethal effect of dendritic cells on neuroblastoma cells: Lethal effect of dendritic cells stimulated T cells in each experimental group ( dendritic cells: neuroblastoma cells=100∶1,50∶1,20∶1 respectively) on neuroblastoma cells was significantly higher than that in control group[(31.00 ±4.41)%,(30.92±5.27)%,(33.57±5.35)%,(26.23±5.20)%, t=3.51,2.98,4.24, P < 0.01); But the lethal effect of dendritic cells on neuroblastoma was significantly lower when their ratio was 100∶1 and 50∶1 in comparison with 20:1 (t=2.01,2.36, P < 0.05), and no significant difference in lethal effect existed between the ratio at 100∶1 and 50∶1(t=0.06,P > 0.05). CONCLUSION: Dendritic cells differentiated from human umbilical cord blood mononuclear cells after in vitro induction of cytokines can promote the lethal effect of T cells on neuroblastoma cells. The lethal effect is associated with the concentration of dendritic cells within some range.
BACKGROUND: Dendritic cell is the most major antigen presenting cell of organism. It is proved in recent studies that human umbilical cord blood mononuclear cells induced and cultured in vitro by recombinant human granulocyte-macrophage colony stimulating factor (rhG-MCSF) and recombinant human interleukin -4 (rhIL-4) can generate a great many dendritic cells and promote the lethal effect of T cells on human neuroblastoma, but it is unclear that the lethal effect is associated with the most proper concentration of dendritic cells. OBJECTIVE: To investigate the lethal effect of human umbilical cord blood mononuclear cells induced in vitro by cytokines differentiating into dendritic cells on human neuroblastoma, and its best concentration range. DESIGN: Open experiment. SETTING: Department of Pediatrics, the Medical School Hospital of Qingdao University. MATERIALS: The study was carried out in the Shandong Provincial Key Laboratory (Laboratory for the Department of Pediatrics of th e Medical School Hospital of Qingdao University) during September 2005 to May 2006. Human umbilical cord blood samples were taken from the healthy newborn infants of full-term normal delivery during October to November 2005 in the Medical School Hospital of Qingdao University, and were were voluntarily Main reagents: neuroblastoma cell line SK-N-SH (Shanghai Institute of Life Science, Chinese) donated by the puerperas. Main instruments: type 3111 CO2 incubator (Forma Scientific, USA) Academy of Sciences), RPMI-1640 culture fluid and fetal bovine serum (Hyclone), rhIL-4 (Promega, USA), rhG-MCSF (Harbin Pharmaceutic Group Bioengineering Co. Ltd.), rat anti-human CD1a monoclonal antibody and FITC- labeled rabbit anti-rat IgG (Xiehe Stem cell Gene Engineering Co. Ltd.). METHODS: ① Human umbilical cord blood mononuclear cells obtained with attachment methods differentiated into human umbilical cord blood dendritic cells, presenting typical morphology of dendrit iccells after in vitro induction by rhG-MCSF and rhIL-4. (2) Different concentrations of dendritic cells [dendritic cells: neuroblastoma cells = 20: 1, 50: 1, 100: 1 , 1 × 109 L-1)], 1 × 109 L-1 T cells and 1 × 107 L-1 neuroblastoma cells were added in the experimental group. 1 × 109 L-1 T cells and 1 × 107 L-1 neuroblastoma cells were added in the control group. ③ Main surface marker CD1a molecules of dendritic cells were detected with indirect immunofluorescence, and the percent rate of dendritic cells was counted with ultraviolet light and expressed as the expression rate of CD1a + cells. ④ Single effector cells and target cells were separately set in the experimental group and control group to obtain the lethal effect. The lethal effect of dendritic cells on neuroblastoma cells are indirectly evaluated by detecting cellular survival with MTT assay. The lethal effect (%) = (1-A experimental well-A effector cell well / A target cell well × 100% .⑤The experimental data we MAIN OUTCOME MEASURES: ① Morphological characters of dendritic cells in the process of induction and differentiation. ② CD1a + cellular expression rate. ③ Lethal effect of dendritic cells on neuroblastoma cells. RESULTS: ① Morphological characters of dendritic cells in the process of induction and differentiation: On the 15th day after human umbilical cord blood mononuclear cells were induced by rhG-MCSF and rhIL-4, typical morphology of dendritic cells could be seen under an inverted microscope. ②Expression rate of CD1a + (43.12 ± 5.83)%. (3) Lethal effect of dendritic cells on neuroblastoma cells: Lethal effect of dendritic cells stimulated T cells in each experimental group (dendritic cells: neuroblastoma cells = 100: 1, 50: 1, 20: 1 respectively) (31.00 ± 4.41)%, (30.92 ± 5.27)%, (33.57 ± 5.35)%, (26.23 ± 5.20)%, t = 3.51, 2.98, 4.24, P < 0.01); But t he lethal effect of dendritic cells on neuroblastoma was significantly lower when their ratio was 100: 1 and 50: 1 in comparison with 20: 1 (t = 2.01, 2.36, P <0.05) : 1 (t = 0.06, P> 0.05) CONCLUSION: Dendritic cells differentiated from human umbilical cord blood mononuclear cells after in vitro induction of cytokines can promote the lethal effect of T cells on neuroblastoma cells. The lethal effect is associated with the concentration of dendritic cells within some range.