| ObjectiveTo investigate the effects of dose-dependent and time-dependent of endotoxin,also known as lipopolysaccharide(LPS),on the proliferation,cell cycle ratio,cell surface marker levels,osteogenic differentiation capacity and cytokine expression of human umbilical cord mesenchymal stem cells(h UC-MSCs),in order to further understanding the functional roles and values of intestinal microbiome metabolites such as endotoxin.Methods1.By CCK-8(Cell Counting Kit-8)cell proliferation technique,the h UC-MSCs were cultured in stem cell medium containing ten different concentrations with LPS(0.098,0.195,0.39,0.78,1.56,3.12,6.25,12.5,25,50EU/m L),and cell proliferation was measured at 24,48 and 72 h after culture.2.Using flow cytometry,the h UC-MSCs were cultured in stem cell medium containing three different concentrations with LPS(0.098,1.56 and 25EU/m L)and the cell cycle was examined at 24,48 and 72 h after culture,respectively.3.Using flow cytometry,the h UC-MSCs were cultured with stem cell medium containing three different concentrations with LPS(0.098,1.56 and 25EU/m L),and the expression of positive cell surface markers(CD90,CD105,CD73)and negative cell surface markers(CD45,CD34,CD19,CD11 b,HLA-DR)were detected at 24,48 and72 h after culture,respectively.4.Using the colorimetric method for osteogenic differentiation potential of stem cells,the h UC-MSCs were cultured in osteogenic differentiation medium containing three different concentrations with LPS(0.098,1.56 and 25EU/m L)to induce osteogenic differentiation of cells and stained with alizarin red S.5.By enzyme linked immunosorbent assay(ELISA),the h UC-MSCs were cultured in stem cell medium containing five different concentrations with LPS(0.098,0.39,1.56,6.25,25EU/m L)for 24,48 and 72 h.The cell culture supernatants were collected and the expression of cytokines IL-6 and IFN-γ were detected.Results1.The effect of LPS on the proliferation of h UC-MSCs: when LPS is cultured for24 h,the overall inhibition of cell proliferation is observed,and the inhibition is most significant at the concentration of 0.78EU/m L with LPS,and the inhibition of proliferation tend to diminish as the LPS concentration decreased or increased.The inhibition of cell proliferation is generally reduced when LPS is cultured for 48 h.When LPS is cultured for 72 h,it shows a tendency to promote cell proliferation,and the promotion effect is most significant at low concentrations,while the promotion effect is weakened when the concentration increased.2.The effect of LPS on the cell cycle of h UC-MSCs: when LPS is cultured for24 h,the percentage of G2/M phase increased in the low concentration group,the percentage of S phase increased in the medium concentration group,and the percentage of S and G2/M phases increased in the high concentration group.When LPS is cultured for 48 h,the proportion of S and G2/M phases increased in the medium concentration group.When LPS is cultured for 72 h,the S and G2/M phases increased in the low concentration group and the S phase increased in the medium concentration group as well as in the high concentration group.3.The effect of LPS on the cell surface markers of h UC-MSCs: the effect on CD90 expression is slightly reduced at 48 h in medium concentrations with LPS stimulated cells,while the remaining concentrations and time points of action shows no significant differences.For CD105,CD73 and CD45,CD34,CD19,CD11 b,HLA-DR molecules,there are no differences between the concentration groups and the control group at 24,48 and 72 h after culture.4.Effect of LPS on osteogenic differentiation of h UC-MSCs: by Alizarin Red S staining,the blank control group and negative control group of the three concentrations with LPS show negative staining,the positive control group and the three concentrations with LPS group as well show bone calcium nodules,the staining results show positive,and there is no difference in the comparison with the positive control group as observed under the microscope.5.Effect of LPS on the expression of secreted cytokines in h UC-MSCs: 1)When LPS is cultured for 24 and 48 h,IL-6 expression is reduced in the 0.098 and 0.39 EU/m L concentration groups compared with the control group,and no statistical significance can be found in the 1.56,6.25 and 25EU/m L concentration groups compared with the control group,and when LPS is cultured for 72 h,all five concentration groups compared with the control group show the data are not statistically significant.2)The expression of IFN-γ in the culture supernatant of h UC-MSCs is not detected with or without LPS.Conclusions1.The influence of LPS on the proliferation of h UC-MSCs reflects a combination of dose-effect and time-efficiency of LPS.When the effect of time-dependent is short,overall cell proliferation is inhibited,when the effect of time-dependent is prolonged,low concentrations of LPS promote cell proliferation.2.Different dose-dependent and time-dependent of LPS resulted in different degrees of cell cycle arrest in h UC-MSCs.3.The expression of surface antigenic molecules of h UC-MSCs is not affected by different dose-dependent and time-dependent of LPS.4.LPS can induce osteogenic differentiation of h UC-MSCs,but the osteogenic differentiation ability is not significantly related to the dose-effect and time-efficiency of LPS.5.Different dose-dependent and time-dependent of LPS result in different levels of IL-6 secretion by h UC-MSCs,and IFN-γ secretion by h UC-MSCs is not confirmed. |
PDF Full Text Request