| Objective:To investigate the effects of hUC-MSCs on the proliferation of CIK cells,the proportion of cell subpopulations,the expression of immune factors and the cytotoxicity,and the related mechanisms.Methods:The hUC-MSCs isolated and identified in our laboratory in the previous period were recovered to obtain adherent cells and the morphology was observed.Peripheral blood mononuclear cells(PBMCs)were extracted by conventional methods.After the CIK cells were induced to expand,the morphological changes of the cells were observed,cell proliferation was detected by the CCK-8 method,and the expression of the cell surface antigen was detected by flow cytometry.The experiment was divided into two parts:Experiment 1 and Experiment 2.Experiment 1(direct contact co-cultivation):hUC-MSCs were seeded in culture flasks.After hUC-MSCs was adhered,the culture medium was removed and CIK cells were added.Four groups were set up:Group A(1×10~4/ml MSCs+5×10~5/ml CIK),Group B(5×10~4/ml MSCs+5×10~5/ml CIK),Group C(1×10~5/ml MSCs+5×10~5/ml CIK),Group D(control group,5×10~5/ml CIK).Experiment 2(indirect contact co-cultivation):hUC-MSCs were seeded in the bottom plate of Transwell culture wells.After the hUC-MSCs were adherent,the culture medium was removed and the cells were transferred with CIK cells in a small chamber.Four groups were set up:Group a(1×10~4/ml MSCs+5×10~5/ml CIK),Group b(5×10~4/ml MSCs+5×10~5/ml CIK),Group c(1×10~5/ml MSCs+5×10~5/ml CIK),Group d(control group,5×10~5/ml CIK).The cell proliferation was measured by counting the number of CIK cells in each group.The expression of CD3,CD4,CD8,CD56,and CD16 on the surface of CIK cells in each group was detected by flow cytometry.The levels of IL-10,IL-2,PGE2,TGF-?,TNF-?,and IFN-?in the supernatant of each group were detected by ELISA and the ability of each group of CIK cells anti-leukemia cells was detected by CCK-8 method.Results:After resuscitation and passage thehUC-MSCs began to adhere to the wall after 12 to 48 hours.The cells showed a long spindle shape,similar to fibroblast-like cells,vortex-like or unipolar to radial growth.PBMCs began to form colony on the fifth day after induction.The number of PBMCs increased gradually after amplification,and the volume gradually increased.The shape was irregular and the size was uniform.CIK cells were cultured for 14 days and the result of flow cytometry showed:CD3~+CD4~+cells accounted for29.5%of the total cells,CD3~+CD8~+cells accounted for 50.9%of the total cells,CD3~+CD56~+cells accounted for 13.2%of the total cells.CD3~-CD56~+CD16~+cells accounted for 6.9%of the total cells.Experiment 1 Results:hUC-MSCs can promote the proliferation of CIK cells through direct contact with cells.HUC-MSCs can up-regulate the ratio of CD3~+CD8~+,CD3~-CD56~+CD16~+and CD3~+CD56~+in the subset of CIK cells;increase the expression of cytokines of IFN-?,TNF-?and IL-2.The expression of IL-10 and PGE2 decreased;enhances the anti-leukemic effect of CIK cells.And the above results have a certain concentration correlation.Experiment 2 Results:hUC-MSCs can inhibit CIK cell proliferation through indirect factors;Down-regulate the ratio of CD3~+CD8~+,CD3~-CD56~+CD16~+and CD3~+CD56~+in CIK cell subsets;increase the expression of cytokine IL-10;decrease the expression of TNF-?;inhibition of CIK cells anti leukemic cells.And the above results have a certain concentration correlation.Conclusions:The induction and expansion of PBMC in vitro can be used as a source of donor cells for clinical treatment of CIK cells.HUC-MSCs can regulate the immune activity of CIK cells through cell contact and secretion-related soluble cytokines,and this regulatory effect has a certain correlation with the number of hUC-MSCs. |
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