| Objective:Human umbilical cord mesenchymal stem cells(hUC-MSC)were isolated from neonatal umbilical cord in vitro to detect the effects of different concentrations of metformin(METF)medium on cell morphology,cell proliferation,cell surface marker expression and cell cycle.Methods:The hUC-MSCs were isolated from the umbilical cord of healthy full-term newborns for subculture,and the cells were identified by the third generation upflow cytometry.When the cells were cultured to the 7th passage,culture medium(0 mM,0.1 mM,1 mM,5 mM,10 mM,20 mM)containing different concentrations of metformin was added,and the morphology of the cells was observed under an inverted microscope,and photographed by MTT colorimetry.The effects of medium containing different drug concentrations on cell proliferation were detected at 24,48,and 72 hours,respectively.At the same time,hUC-MSCs co-cultured with the drug for 24 hours were analyzed by flow cytometry.After 48 hours,the expression of cell surface markers was analyzed.The proportion of cell cycle in each group was analyzed.Results:1.Morphological analysis of human umbilical cord mesenchymal stem cells:when the concentration of metformin was0.1mM,1mM,the cell morphology did not change significantly.When the drug concentration was 5~20mM,the cell morphology increased with the drug concentration and the culture time prolonged,so that the morphology changed the more remarkable.2.MTT colorimetric assay for the relative proliferation rate of each group of cells:METF=0.1mM:24h 101.28±0.98,48h104.06±1.76,72 h 101.51±0.67;METF=1mM:24h 116.03±1.82,48 h 84.20±1.60,72h70.98±2.47;METF=5mM:24h 115.13±3.06,48 h 64.64±1.20,72 h 43.83±2.38;METF=10mM:24h 108.37±1.26,48 h 59.69±1.90,72 h 25.58±0.61;METF=20mM: 24h86.64±0.66,48 h 58.38±2.52,72 h 17.75±1.35;According to the experimental results,it can be seen that the concentration of metformin is 0.1mM,which can promote the proliferation of hUC-MSC.The drug concentration of 1-10 mM can increase the proliferation rate of hUC-MSC in the early stage of culture.With the prolongation of culture time,cell proliferation is gradually inhibited.The concentration of metformin was 20 mM to inhibit cellproliferation,and the inhibition increased with time(P<0.05).3.The effect of flow cytometry on the expression of cell surface markers in each group:METF=0mM:CD44 0.75±0.18,CD9096.96±0.44,CD105 72.80±2.90;METF=0.1mM: CD440.53±0.25,CD90 96.93±0.89,CD10576.75±2.15;METF=1mM:CD440.53±0.08,CD90 97.54±0.22,CD105 72.88±2.37;METF=5mM:CD44 0.50±0.24,CD90 97.21±0.47,CD105 66.52±2.70;METF=10mM:CD44 0.60±0.14,CD90 97.47±0.31,CD105 66.74±4.33;METF=20mM: CD44 0.62±0.28,CD90 97.67±0.22,CD105 57.25±6.92.According to the experimental results,when the concentration of metformin was 5mM,10 mM,20mM,the expression of CD105 gradually decreased with the increase of drug concentration(P<0.05).When the concentration of metformin was 0.1mM-20 mM,it did not affect CD44 and CD90.4.The effect of flow cytometry on the cell cycle of each group of cells:METF=0mM:G0/G1(%)66.40±2.65,S(%)29.03±3.19,G2/M(%)3.56±2.20;METF=0.1mM:G0/G1(%)64.16±1.20,S(%)29.47±1.00,G2/M(%)6.37±0.40;METF=1mM:G0/G1(%)72.89±1.14,S(%)24.19±2.16,G2/M(%)2.34±1.02;METF=5mM:G0/G1(%)73.59±1.58,S(%)20.92±0.77,G2/M(%)5.48±0.84;METF=10mM:G0/G1(%)77.55±1.25,S(%)16.32±1.10,G2/M(%)6.18±1.01;METF=20mM:G0/G1(%)83.85±1.68,S(%)13.68±1.67,G2/M(%)2.47±0.23;According to the experimental results,the concentration of metformin can decrease the proportion of G0/G1 phase(P<0.05),which can promote the proliferation of hUC-MSC.With the increase of drug concentration,cell proliferation is gradually inhibited.Conclusions :The concentration of metformin at 0.1mM can promote the proliferation of human umbilical cord mesenchymal stem cells,and inhibit the proliferation of human umbilical cord mesenchymal stem cells and the expression of surface marker CD105 at concentrations of 5~20mM.Different concentrations of metformin are not related to CD44.The expression of CD90 has an effect. |
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