| Objective: To investigate the effect of shenfu injection(SFI) with cytokines on induction and function of dendritic cells(DCs) drived from the human cord blood mononuclear cells (CBMNCs) and the acute myeloid leukemia M2 cell line HL-60 cells in vitro.Methods: In vitro, cord blood mononuclear cells from term fetus′cord and HL-60 cells were cultured with different concentrations SFI whose final concentrations were 9.38mg/ml, 18.75mg/ml, 37.5mg/ml,75mg/ml and 150mg/ml , or only with Granulocyte macrophage colony stimulating factor (GM–CSF) plused interleukin-4 (IL-4), or GM-CSF+IL-4 with above different concentrations SFI. The morphologic changes were observed with optical invert microscope and Wright staining. The expression rate of CD83, CD1a, CD80 and HLA-DR on DCs were detected by flow cytometry (FCM). The capability of DCs stimulating allo-lymphocyte proliferation was tested with mixed leukocyte reaction (MLR) by MTT. The level of INF-γin supernatant was measured by ELISA method.Results 1. The morphologic changes of cells: Pre-treat HL-60 cells and freshly isolated CBMNCs appeared as dispersed, spherical with a smooth surface morphology. 2 days after cultured, some cells of the two groups cultured only with GM-CSF+IL-4 appeared large size, multiply and began to show anomalo-shape. 8~12 days past, there was a significant population of cells with typical dendritic cells morphological features. While HL-60 cells and CBMNCs cultured with different concentrations of SFI developed bidirectional changes: 150mg/ml and 75mg/ml SFI induced all cells to death; 37.5mg/ml SFI led to slow proliferation; 18.75mg/ml and 9.38mg/ml SFI increased cell size and proliferated obviously. 8~12 days after cultured, there were no dendritic-like cells. In addition, HL-60 cells and CBMNCs co-cultured with GM-CSF+IL-4 and different concentrations of SFI developed bidirectional changes. 150mg/ml SFI groups leaded all cells to death. 75mg/ml SFI groups increased slightly in cell size and proliferated slowly and only few dendritic-like cells could be found. However, 8~12 days past, an increase in cell size and the rate of typical dendritic in morphology was observed in cultured with 37.5mg/ml, 18.75mg/ml, 9.38mg/ml SFI associating with GM-CSF+IL-4. 18.75mg/ml SFI groups in HL-60 cells and 37.5mg/ml SFI groups in CBMNCs were the highest proportional groups of dendritic-like cells among them. In addition, grape-like clusters could be found.2. Phenotype of cultured cells: The expressions of DCs markers CD83, CD1a, HLA-DR and CD80 displayed very weak on pre-treat HL-60 and freshly isolated CBMNCs and the control groups of them. Both HL-60 cells and CBMNCs cultured only with SFI expressed lowly as well. Both HL-60-DCs and CBMNCs-DCs expressed higher levels of these phenotypes after co-cultured with GM-CSF+IL-4 for 8~12 days compared with untreated and only SFI cultured groups (the expressions of HL-60-DCs were 8.33%±1.76%,11.86%±3.86%,7.70%±1.51% and 1.82%±0.27%. CBMNC-DCs were 12.36%±2.79%,15.23%±2.04%,26.90%±6.17% and 14.6%±4.31%, respectively) (p<0.05,exclude CD80 on HL-60-DCs,HLA-DR on CBMNC-DCs). Treatments of cells with GM-CSF+IL-4 and different concentrations SFI resulted in different changes of these phenotypes. Compared with other groups'cells, HL-60 cells cultured with 18.75mg/ml SFI+GM-CSF+IL-4 and CBMNCs cultured with 37.5mg/ml SFI+GM-CSF+IL-4 expressed the significantly higher levels DCs markers (p<0.05) (HL-60-DCs were CD83 18.75%±6.18%, CD1a 24.32%±7.44%, HLA-DR 26.99%±7.42% and CD80 18.24%±3.13%. CBMNC-DCs were 27.33%±8.26%, 39.28%±9.41%, 49.43%±15.89% and 32.07%±5.92%, respectively). In addition, they were observed good synergistic reactions (p<0.05). There were no significant differences of phenotypic expressions between cells cultured with other concentrations SFI plus GM-CSF+IL-4 and with GM-CSF+IL-4,in spite of the former was superior to the latter (p>0.05). There was significant difference in expression rate of HLA-DR and CD80 (CBMNCs groups superior to HL-60 cells groups, p<0.01), while there was no noticeable diversity of CD83 and CD1a between HL-60 groups and CBMNCs groups (p>0.05).3. Ability of stimulating T lymphocyte proliferation: There was weak ability of stimulating T lymphocyte proliferation in the control groups, and the groups cultured with 18.75mg/ml SFI of HL-60 cells and CBMNCs. The stimulated indexs did not elevated accompany with the gradually increasing E:T ratio (p>0.05). In contrast, the DCs induced with GM-CSF+IL-4 or SFI+GM-CSF+IL-4 elicited significant stronger abilities of stimulating T lymphocyte proliferation than the others .When E:T ratios ranging from 1:100, 1:30, 1:10, the stimulated indexs are HL-60-DCs 1.606±0.074 vs. 2.280±0.097 vs. 2.415±0.090 and 2.106±0.174 vs. 2.728±0.125 vs. 3.196±0.137,CBMNC-DCs 1.827±0.129 vs. 2.217±0.157 vs. 2.503±0.159 and 2.218±0.160 vs. 2.632±0.079 vs. 3.143±0.043 respectively, (all r=0.949,P<0.01). Furthermore, there was significant difference between these two groups respectively (p<0.05). In addition, there was no noticeable differentce in the ability of stimulating T lymphocyte proliferation between HL-60-DCs and CBMNC-DCs (P>0.05).4. The examination of INF-γlevel: Different levels of INF-γwere detected in the supernatant of culturing HL-60 cells and CBMNCs in different culturing conditions. INF-γlevels of cells cultured with 18.75mg/ml SFI were obviously lower than with GM-CSF+IL-4 [HL-60 (22.887±7.575) pg/ml vs. (37.350±8.306) pg/ml, CBMNCs (30.630±6.993) pg/ml vs. (48.020±9.280) pg/ml, p<0.01]. Contrast to GM-CSF+IL-4 plused other concentrations SFI groups, INF-γlevels were significantly higher in HL-60 cells cultured with 18.75mg/ml SFI and CBMNCs with 37.5mg/ml SFI [ (73.340±14.402) pg/ml, (90.410±12.189) pg/ml, respectively, p<0.01]. In addition, there was observed good synergistic reactions (p<0.05). The other groups with different concentration SFI plus GM-CSF+IL-4 had differently increasing levels of INF-γcompared with only with GM-CSF+IL-4, but there was no statistical significances. Furthermore, HL-60 groups and CBMNCs groups secrete the similar levels of INF-γ(p>0.05).Conclusions1. In vitro, cultured only with different concentrations of SFI could not derive from CBMNCs and HL-60 cells to DCs.2. In vitro, DCs could be derived from HL-60 cells and CBMNCs co-cultured with GM-CSF and IL-4. The DCs derived from CBMNCs and leukemia cells could express DCs-specific phenotype and function, but their surface markers and function were unmature.3. In vitro, appropriate concentrations of SFI plus GM-CSF+IL-4 could induce differentiation and maturation of HL-60-DCs and CBMNC-DCs, promote maturation of DCs and enhance their immune stimulatory function. 4. The optimal concentration of SFI is 18.75mg/ml or 37.5mg/ml respectively which plus GM-CSF+IL-4 could induce HL-60 cells or CBMNCs into DCs in vitro. In addition, these concentrations SFI and GM-CSF+IL-4 were observed good synergistic reactions.5. In a certain range, different concentration of SFI has bidirectional regulating action which inhibits or encourages propagation of human CBMNCs and AML HL-60 cells.6. The ability of antigen presenting of CBMNC-DCs is similar to HL-60-DCs.7. Maturated DCs secrete INF-γand their ability of antigen presenting increased correspondently. |
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