| Objective: By neonatal umbilical cord isolated between umbilical cord mesenchymal stem cells in vitro h UC-MSC(Human umbilical cord mesenchymal stem cells,h UC-MSC),then the induction of neural stem cells and to detect the different concentrations of metformin ectomesenchymal stem cell source for neural stem cell Proliferation,activity and the influence of the cell cycle.Methods: Sterile conditions in vitro isolated from healthy full-term newborn umbilical cord h UC-MSC,will the subculture,instrument to third-generation upflow cells to identify cells,then take three generations in the logarithmic growth of umbilical cord mesenchymal stem cells and the induction of neural stem cells,ectomesenchymal stem cells by flow cytometry instrument to identify sources of neural stem cells.Will induce neural stem cells suspension inoculation to 24 hole culture plate,and each group of six holes,stay 3 days later,after adding metformin makes its final concentration respectively0 5 uM uM,15,10 uM,uM,20 50 uM,uM,continue to develop,in 24,48,72 hours after enzyme standard to test it at 450 nm absorbance value(OD value).The proliferation rate and activity of induced neural stem cells in different concentrations of metformin were calculated.At the same time,flow cytometry was used to detect the effect of metformin at different concentrations and at different times on the induced neural stem cell cycle.Then SPSS21.0 statistical software was used for statistical analysis.Results :(1)The Proliferation rates of METF-1,METF-2,METF-3,METF-4 and METF-5 groups after co-culture with induced neural stem cells at different concentrations for 24 h were as follows:(128.68±0.58)%,(136.33±1.53)%,(158.86±1.48)%,(137.00±2.00)%,(112.33±0.58)%,the differences between the experimental group and the blank control grou P and among the experimental groups were statistically significant(P<0.001),indicating that the proliferation of neural stem cells derived from mesenchymal stem cells was promoted in each culture group after 24 h,and the Proliferation rate was the highest when METF concentration was 15umol/L;Gradually with the extension of incubation timetogether,cell proliferation inhibition,is affected by its concentration,a total of 48 h after training drug concentration for 20 umol/L,the proliferation rate was 110.33±0.58,it is still between the source of mesenchymal stem cells can promote neural stem cell proliferation,and METF-1,METF-2,METF-3,METF-5 groups of Proliferation rate(89.33±0.58)%,(93.67±0.58)%,(96.00±0.00)%,(90.33±0.58)%,In other words,when the drug concentration was 5,10,15 and 50umol/L after 48 h co-culture,the cell proliferation was inhibited,and the proliferation rate of each group was statistically significant(P<0.001).Over time,continue to develop 72 h after cell proliferation inhibition is more significant,METF-1,METF-2,METF-3,METF-5 groups of proliferation rate(84.33±0.58)%,(89.67±1.16)%,(93.00±0.00)%,(87.33±1.16)%,however in 20 umol/L drug concentration(proliferation rate was 105.67±-1.16)still can promote cell proliferation,but its proliferation rate declined with the extension of incubation time,statistically significant difference(P <0.001).(2)After co-culture of mesenchymal stem cell derived neural stem cells with different concentrations of METF for 24 h,the cell cycle G0/G1 phase accounted for(91.62±0.66)%in the blank control group,and the cell cycle G0/G1 phase accounted for(90.59±0.41)%,(89.39±0.72)%,(83.81±0.43)%,(85.92±0.79)% and(86.97±0.15)% in the metf-1,metf-2,metf-3,metf-4 and metf-5 groups,respectively.The difference between the experimental group and the blank control group and between the experimental groups was statistically significant(P<0.001).Compared with the control group,the proportion of G0/G1 phase in each experimental group was reduced and cell proliferation was promoted.Moreover,when METF concentration was at 15umol/L,the proportion of G0/G1 phase(83.81±0.43)was the lowest and the proliferation index was the highest(16.19±0.43).After 48 h of co-culture,the G0/G1 phase of cell cycle in the blank control group accounted for(91.18±0.49)%.The proportion of G0/G1 phase of induced neural stem cells increased at different METF drug concentrations.METF-1,2,METF METF--3,cell cycle,METF METF-4-5 group G0 /G1 phase ratio(94.41 + 0.39)%,respectively(93.27 + 0.37)%,(92.18 + 0.19)%,(88.08 +0.55)%,(93.85 + 0.45)%,the experimental group and the blank control group and the comparison between the experimental group,the difference is statistically significant(P <0.001),suggesting that cell proliferation inhibited gradually,and with the increase of drug concentration,increase in G0 / G1 phase proportion is not obvious,When the concentration of the drug was 20umol/L,the ratio of G0/G1 phase(88.08±0.55)was reduced compared with the control group,and the ratio of G0/G1 phase(93.85±0.45)was significantly increased when the concentration of the drug was 50umol/L,and the ratio of G0/G1 phase(93.85±0.45)was significantly increased compared with that of the control group.Conclusion: Metformin concentration and action time of ectomesenchymal stem cell source for neural stem cells has interaction,24 h,between different concentrations of metformin on the source of mesenchymal stem cells promotes proliferation effects of neural stem cells,the drug concentration of 15 umol/L promote the strongest,with the extension of action time,48,72 h,different concentrations of metformin ectomesenchymal stem cell source for neural stem cells showed inhibition of proliferation effects,and in the drug concentration of 20 umol/L,to induce neural stem cells is to promote the proliferation.Above all,metformin in a certain dose concentration range and time between the source of mesenchymal stem cells proliferation effects of neural stem cells,this experiment suggests the metformin to promote the proliferation of neural stem cells induced by type,it can be transplantation for the treatment of diseases such as nerve damage,but these should pass more clinical experiments to determine the scope of the scope of its safety,effective and best treatment range,to benefit more patients. |
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