| Objective: to investigate the effects of shenfu injection(SFI) on the induction of chronic myeloid leukemia cells(CML)and chronic myeloid leukemia cell line K-562 cells into dendritic cells(DCs). Method: In vitro, peripheral blood mononuclear cells from CML patients and K-562 cells were cultured with different concentrations SFI whose final concentrations were low concentration (9.38mg/ml), middle concentration (37.5mg/ml, 75mg/ml) and high concentration (150mg/ml, 300mg/ml), Granulocyte-macrophage colony-stimulating factor (GM–CSF) /interleukin-4 (IL-4) and GM-CSF/IL-4 combined with different concentrations of SFI respectively. The morphologic changes were analyzed with optical invert microscope. The phenotype of DCs were detected by flow cytometry (FCM) with CD1a, CD80, CD83 and HLA-DR momoclonal antibodies. The capability of stimulating allo-lymphocyte proliferation was tested with mixed leukocyte reaction (MLR). The level of IL-12 in supernatant was measured by ELISA method. Results 1. The morphologic changes of cells. Freshly K-562 and CML cells appeared as dispersed, spherical with a smooth surface morphology. After days of culture, some cells cultured with GM-CSF/IL-4 appeared larger and began to show anomalo-shape. 14 days past, there was a significant population of cells with dendritic morphologiy. Yet, K-562 and CML cells cultured in different concentrations of SFI developed different changes. High concentration SFI induced all cells to death or slowly proliferation. Both middle concentration SFI and low concentration SFI increased cells size and proliferation noticably. After 14 days of culture, however, dendritic-like cells were not found. In addition, K-562 and CML cells co-cultured with different concentrations of SFI combined with GM-CSF/IL-4 developed different changes. High concentration SFI induced all cells to death or no significant changes in size and proliferation. After 14 days of culture, only a few dendritic-like cells could be found. However, between days 10 and 12, cultures with GM-CSF, IL-4 and middle concentration SFI or GM-CSF/IL-4 plus low concentration SFI consistently displayed an increase in cell size and showed grape-like clusters with dendritic projections in the predominant population. 2. Phenotype of cultured K-562 and CML cells. Freshly K-562 and CML cells displayed very weak expressions of CD1a, CD80, CD83 and HLA-DR. In contrast, cells co-cultured with GM-CSF/IL-4 expressed high levels of these phenotypes on day 14(the expressions of K-562DCs were 13.01%±3.19%, 12.37%±3.79%, 15.40%±3.98% and 14.89%±3.45%. CML-DCswere 12.93±3.52%, 12.37%±3.79 %, 15.40%±3.98% and 14.22%±3.81%), cells only cultured with SFI expressed low levels of CD1a, CD80, CD83 and HLA-DR. Treatment of cells with GM-CSF/IL-4 and different concentrations SFI resulted in different changes of these phenotypes. Those co-cultured with high concentration SFI expressed much lower levels of CD1a, CD80, CD83 and HLA-DR than those grown in GM-CSF/IL-4 with middle concentration SFI or low concentration SFI (p<0.01). In contrast, the expressions of CD1a, CD80, CD83 and HLA-DR by CML-DCs and K-562DCs were enhanced when they were cultured with middle concentration SFI plus GM-CSF/IL-4. Compared with other groups'cells, cells cultured with 75mg/ml SFI/GM-CSF/IL-4 expressed the highest levels phenotypes (K-562DCs were 26.62%±4.02%, 28.00%±3.42%, 42.09%±3.95% and 34.65%±10.49%. CML-DCs were 25.24% ±4.52%, 24.67% ±4.32%, 37.81% ±5.00% and 32.46%±5.06%). There were no significant differences of phenotypic expressions between cells cultured with low concentration SFI plus GM-CSF/IL-4 or with GM-CSF/IL-4. Similar results were obtained by K-562 and CML groups. 3. Ability of Stimulating T lymphocyte proliferation. K-562 cells, CML cells and cells cultured with 75mg/ml SFI stimulated T lymphocyte proliferation weakly. According to responder ratios ranging from 1:50, 1:10 to 1:2, cells cultured with GM-CSF/IL-4 or SFI/GM-CSF/IL-4 elicited significantly strong abilities of stimulating T lymphocyte proliferation.
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