The Screening Of Signaling Pathways In Osteoblast And The Effect Of PI3K/Akt Signaling Pathway On The Proliferation Of MC3T3-E1Cells

Objective:This study aimed to inhibit the signaling molecules in MC3T3-E1osteoblasts, screening the signaling pathways that affect the proliferative activity of the MC3T3-E1cells, and verify its effectiveness.Methods:Using44inhibitors treated MC3T3-E1cells respectively, detect the cell activity by MTS assay, and screening the signaling molecules affected the proliferative activity of the MC3T3-E1cells. We choose a signaling molecule from screened molecules for further study. The cells treated with the corresponding inhibitor, and the validity is tested. Fistly, we added the inhibitor to MC3T3-E1cells,and measured the effect of cell cycle via FACS analysis. Next, we detected the expression level of relevant proteins in cell after treated with inhibitor by western blot analysis. We selected Akt signaling molecule for further research.Results:Using44inhibitors treated MC3T3-E1cells, demonstrated that7inhibitors could significantly restrain proliferative activity of the MC3T3-E1cells, include IGF-1R/INSR/ALK、AKT、SRC/ABL、KIT. PI3K、MET/VEGFR and mTOR. Inhibition of Akt signaling using PF-04691520(S2743) which is pharmacologic inhibitors of Akt, finding that the cells in G1phase increased obviously and the cells in S, G2/M phase descreaded by FACS analysis compared to control group. The results indicate the cell cycle is blocked and the cell proliferation is attenuated Furthermore, western blot analysis showed that the level of p-Akt and CCND1are descreaded in MC3T3-E1cells of treated with PF-04691520. The consequence suggested the cell cycle is suppressed, and the negative effect is associated to decrease the level of phosphorylation Akt.Conclusion:These findings indicate that several signaling pathways affect the proliferation of osteoblasts,and the PI3K/Akt signaling pathway can promote the proliferation of osteoblasts.