Mechano-Growth Factor Accelerates The Growth And Osteogenic Differentiation Of Rabbit Mesenchymal Stem Cells Through The PI3K/AKT Pathway

Section 1 Isolation and culture rabbit bone marrow mesenchymal stem cells in vitroObjective:The bone marrow mesenchymal stem cells were obtained from the rabbit.Methods:Under sterile conditions, the bone marrow mesenchymal stem cells were isolated from New Zealand White rabbits in vivo. And observation of cell morphology, and CD44 in Immunofluorescence Assay and identification of bone marrow stromal stem cells. The expression of CD34 and get optimal growth of bone marrow mesenchymal stem cells by MTT method.Results:The rMSCs were observed. The cell density increases with time longer, and eventually covered the entire bottom. CD44 rMSCs has a higher expression however, CD34 rMSCs without expression. Take P2,P3,P4,P5 cultured cells using MTT method, respectively the growth 1-10 days. Cells grew best was in the 4th generation,and significantly better than the other three generations of cells.Conclusion:The rMSCs cells and the optimal generation were obtained.Section 2 Mechano-growth Factor Accelerates the Growth and Osteogenic Differentiation of Rabbit Mesenchymal Stem Cells Through the PI3K/AKT PathwayObjective:Mechano-growth factor (MGF) accelerated the growth and osteogenic differentiation of rMSCs through the PI3K/AKT pathway.Methods:The optimal concentration was selected by MTT. The effect of MGF on the PI3K/AKT pathway in rMSCs was observed by Western blot. And The osteogenic differentiation was detected qPCR and Western blot.Results:The maximum growth rate appeared at an MGF concentration of 45 nM on the fifth day of treatment.The phosphorylation states of AKT and mTOR were examined at 0,4,8,12, and 16 h following treatment on day 5 (Fig 4A). Figure 4B shows that the phosphorylation of AKT and mTOR increased. To further explore the function of MGF, the expression levels of ALP and OCN were examined in the osteoblasts that were formed from the rMSCs. The expression of these two proteins was higher than that for the other groups treated with 45 nM MGF for 4 h on the fifth day of treatmentConclusion:MGF accelerated the growth and osteogenic differentiation of rMSCs through the PI3K/AKT pathway. Section 3 The effect of PI3K/AKT Phosphorylation inhibitor (LY294002) on the growth and differentiation of rMSCsObjective:To study the effect of PI3K/Akt on rMBSCs further, the LY294002 was effected on the rMBCs.Methods:Phosphorylation inhibitor LY294002 on the rMBCs and PI3K/Akt, to choose the best concentration the MTT was used. The phosphorylated AKT expression and phosphorylation of mTOR, after effected by LY294002, was detected by Western blot. The rMSCs growth was detected by MTT. The OCN and ALP expression was detected by Western blot.Results:The PI3K/AKT inhibitor LY294002 with 20μmol/L was used to inhibit PI3K/AKT activity. Following the addition of LY294002, the cells were treated with MGF for 1-10 days, and their growth rates were compared with those of cells that did not receive LY294002 treatment. We found that the phosphorylation of mTOR was decreased compared with that of controls. The inhibition of PI3K/AKT also blocked the ability of MGF at 45 nM to increase the growth rate of rMSCs, even after 10 days of treatment. In addition, the expression levels of ALP and OCN were decreased compared with those in control osteoblasts.Conclusion:MGF promotes the growth and differentiation of rMSCs via the PI3K/AKT pathway...