| ObjectiveThrough investigate the effect of dose-dependent and time-dependent Butyric Acid(BA)on the proliferation,cell cycle,expression of surface markers,osteogenic differentiation ability and cytokine expression of human umbilical cord mesenchymal stem cells(hUC-MSCs),so that we can improve the understanding for the biological activity of BA.Methods(1)The hUC-MSCs was resuscitated,cultured and amplificated in vitro.(2)The hUC-MSCs were cultured in stem cell culture medium containing ten different concentrations of BA(0.02,0.04,0.08,0.16,0.32,0.63,1.25,2.5,5,10 mmol/L)for 24h,48h and 72h to detect the cell proliferation in CCK-8 proliferation experiment at different point-in-time.(3)The hUC-MSCs were cultured for 24h,48h and 72h in stem cell culture medium containing three different concentrations of BA(0.02,0.32,5 mmol/L),and the complete medium without BA was as the control group.The cell cycle and the cell surface antigen molecules were detected by flow cytometry,including MSCs positive expression molecules(CD90,CD105,CD73)and negative expression molecules(CD45,CD34,CD19,CD11b,HLA-DR).(4)The hUC-MSCs were cultured in the osteogenic differentiation medium containing three different concentrations of BA(0.02,0.32,5 mmol/L),the osteogenic differentiation medium without BA were used as the positive control,the osteogenic differentiation are induced and the osteogenic differentiation staining was performed.(5)The hUC-MSCs were cultured in stem cell culture medium containing five different concentrations of BA(0.02,0.08,0.32,1.25,5 mmol/L)for 24h,48h and 72h.The cell culture supernatants were collected and the expression of cytokines IL-6 and IFN-y were detected by ELISA.Results(1)The proliferation of hUC-MSCs is related to both dose-effect and time-efficiency of BA.The absorbance value of hUC-MSCs in the concentration range of 0.02 to 2.5 mmol/L within 24 hours is lower than that of the control group,but it is not statistically significant compared with the control group in the 5 and 10 mmol/L concentration groups.By 48h,the cell absorbance value in the 0.02 to 0.63mmol/L concentration area is higher than that in the control group,and the 1.25mmol/L concentration group is meaningless compared with the control group,while the 2.5 to 10mmol/L concentration area is lower than the control group.When the time reaches 72h,the cell absorbance values of the 0.02 and 0.16mmol/L concentration groups are slightly higher than those of the control group,the 0.04,0.08,0.32 and 0.63mmol/L concentration groups have no statistical significance compared with the control group,while the 1.25 to 10mmol/L concentration area is lower than the control group(2)The effect of BA on the hUC-MSCs cell cycle varies with the concentration and time.At 24h,compared with the control group,there is no statistical significance between the each phase cell of low concentration group and the control group.In addition,compared with the control group,the ratio of both G0/G1 and G2/M phases cells in the moderate and high concentration groups increases and S-phase cells' ratio decreases.At 48h,the low and medium concentration groups has no statistical significance compared with the control group,while the ratio of G0/G1 in the high concentration group decreases compared with the control group,and the S and G2/M's ratio increases.When the time reaches 72h,the low and moderate concentration groups has no statistical significance compared with the control group,while the ratio of G0/G1 phase in the high concentration group decreases compared with the control group,and the S and G2/M's ratio increases.(3)The situation for expression of surface antigen molecules on MSCs:the expression ratio of all detected molecula in MSCs surface antigen is not statistically significant compared with the control group at 24h.The expression ratio of CD 105 and CD73 molecules decreases only in the low concentration group at 48h.Although the expression ratio of CD90,CD 105 and CD73 in the high concentration group has a downward trend,the difference is not statistically significant.All expression rates of detected molecular are not statistically significant compared with the control group at 72h.(4)The situation about induction of osteogenic differentiation of hUC-MSCs:after the cells in the negative control group are stained by alizarin red,red calcium salt deposits can be seen.It indicates that the induction of osteogenic differentiation is successful.The low and moderate concentration groups show the same red bone calcium nodules as the negative control group,while the cells in the high concentration group show no nodular bone calcium deposits,and are only stained into a piece of orange-red,and the color is relatively light.(5)The effect of BA on the secretion of cytokines of hUC-MSCs:? At 24h,the expression's situation of IL-6 in the moderate concentration area increases,and the increase is most obvious when the concentration is 1.25mmol/L.The expression level of IL-6 in the low and high concentration groups is not statistically significant compared with the control group.At 48h,the expression of IL-6 decreases in the low concentration group and increases in the moderate concentration area(1.25mmol/L group)and the high concentration group(5mmol/L).At 72h,the expression of IL-6 in the 0.02 and 0.08mmol/L concentration groups decreases and the difference is significant.The IL-6 expression increases when the concentration is 1.25mmol/L,and showes a downward trend when the concentration is 5mmol/L.But the difference is not statistically significant.? The hUC-MSCs hardly expresses IFN-?.At 24h and 48h,the expression level of IFN-? in each concentration group has no statistically significant compared with the control group.At 72h,the expression levels of the 0.08,0.32,and 1.25 mmol/L concentration groups increase slightly,which are different from the control group's.Conclusion(1)The influence of BA on the proliferation and cell cycle of hUC-MSCs reflects the combined effect of both the dose and time.(2)The low concentration of BA at 48h can reduce the expression rate of CD 105 and CD73 on the surface of hUC-MSCs.BA at a certain concentration range has no obvious effect on the expression of hUC-MSCs surface markers within reasonable time.(3)Qualitative analysis of the low and moderate concentration BA groups has no significant effect on hUC-MSCs osteogenic differentiation,and bone differentiation is inhibited in the high concentration group(4)The expression of IL-6 in hUC-MSCs is related to both dose-effect and time-efficiency of BA,hUC-MSCs hardly express IFN-y,no matter whether it has butyric acid or not.(5)The different effects of the different dose-and time-efficiency of BA on the physiological and pathological functional regulation of hUC-MSCs is different. |
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