ABSTRACT
OBJECTIVE@#To investigate the effect of Notch signaling pathways on differentiation of mouse embryonic stem cells(ESC) into haematopoietic stem cells or haematopoietic progenitors cells(HSC/HPC).@*METHODS@#Mouse embryonic stem cells were proliferated in vitro to form embryoid bodies; the differentiation of embryoid bodies should be induced in vitro, the experiments were divided into BE, control, VEGF, DAPT and VEGF-DAPT groups; HSC/HPC ohenotype: CD117D34Sca1 was detected by flow cytometry; the related gene expression was detected by RT-PCR.@*RESULTS@#The number of VEGF-induced HSC/HPC in VEGF group was significantey higher than that in the control and EB group (P<0.05), suggesting that VEGF promotes ESC differentiation to HSC/HPC; the number of DAPT-induced HSC/HPC in DAPT group was significanty higher than that in the Control and EB groups(P<0.05), suggesting that DAPT promotes ESC differentiation to HSC/HPC; the number of VEGF+DAPT-induced HSC/HPC in VEGF-DAPT group was significantly higher than that in VEGF and DAPT groups(P<0.05), suggesting that DAPT and VEGF play a synergistic role to promote differentiation of ESC into HSC/HPC.@*CONCLUSION@#Notch signal pathway inhibits differentiation of ESC into HSC / HPC by VEGF.
Subject(s)
Animals , Mice , Cell Differentiation , Hematopoietic Stem Cells , Mouse Embryonic Stem Cells , Receptors, Notch , Signal Transduction , Vascular Endothelial Growth Factor AABSTRACT
This study was purposed to investigate the effect of Notch signaling pathway on VEGF promoting the proliferation of rat mesenchymal stem cells (MSC). Rat MSC were cultured in vitro, and the cells in logarithmic growth phase were used for experiments. The inhibitor DAPT was used to block Notch signaling pathway, and the effect of the pathway on VEGF promoting proliferation of MSC was observed. The experiment was divided into 4 groups: control, VEGF, DAPT and VEGF+DAPT. The CCK-8 was used to assay the cells proliferation of each group, while RT-PCR was used to detect the changes of related genes (Notch1, Notch2, Flk-1, Hes-1) at mRNA levels. The results indicated that the cells survival rate MSC in DAPT group and VEGF+DAPT group was low in each time point (24 h, 48 h, 72 h), the cell number decreased, and the cells became rounded. The survival rate of MSC in VEGF group was the highest; the difference of cell survival rate was statistically significant between the groups (P < 0.01); Compared with the control group, the mRNA expression level of Notch1, Notch2 and Flk-1 in VEGF group was raised, while the expression level of Notch1 and Notch2 in DAPT group and VEGF+DAPT group come down, with statistically significant differences (P < 0.05); whereas the mRNA expression level of Hes-1 in VEGF group was down-regulated, but that in DAPT group and VEGF+DAPT group was up-regulated, and the difference was statistically significant (P < 0.05). Flk-1 mRNA level in DAPT group and VEGF+DAPT group was slightly lower, but the difference was not statistically significant (P > 0.05). It is concluded that Notch signaling pathway plays an important role in promoting the proliferation of rat MSC, treated with VEGF, however, the DAPT can weaken this effect.