The Comparison Of Different Concentrations BFGF And SDF-1 On Proliferation And Migration Of ST2 And The Mechanism Of BFGF On ST2

Background and objectives:Bone marrow stromal cells(BMSCs)can differentiate into a wide range of specialized cells.ST2 cells are interstitial cells that are isolated from BC8 mice bone marrow stroma and have the potency to differentiate into osteoblast-like cells under appropriate conditions.The selection of growth factor plays a key role in tissue engineering.Many studies have shown that local application of basic fibroblast growth factor(bFGF)can significantly promote bone regeneration in the fracture healing in animal models and human trails.Basic FGF is essential for BMSC survival,sternness and self-renewal.PI3K/Akt and ERK pathways regulate cell viability,apoptosis and migration in many cell types.Although it has been demonstrated that PI3K/Akt activation by bFGF is relevant for fibroblasts,neuron stem cells,and tumor cells proliferation,its role on BMSC growth and migration remains elusive and neither does the mechanism.In this study we explored the molecular mechanisms involved in the regulation of BMSCs by Akt and ERK.To explore the effects and mechanism of bFGF on the inducing proliferation and chemotaxis in ST2 cells.Materials and Methods:(1)In order to select the most effective doses of bFGF,ST2 cells were treated by different concentration of bFGF(10,20,40,80 and 100 ng/ml,)and SDF-1(50,100,200 and 400 ng/ml,),then cell viability was measured with CCK8 assay after being stimulated with growth factors for 1 day,2 days and 3 days;(2)Transwell and wound healing assays were used to investigate cells migration and chemotaxis;(3)Western-blot was performed to determine the activation of signaling protein including p-AKT and p-Erk level;(4)ST2 cells were pretreated by PI3K inhibitor LY294002 and Erk inhibitor U0126,then the proliferation and migration capacities were evaluated,and so did the expression level of p-AKT and p-Erk.(5)Data were collected and given as the mean±standard deviation(SD)for each experiment.A one-way analysis of variance(ANOVA)was used for data analysis using SPSS(Version 20.0).For each test,p<0.05 was considered statistically significant.Results:(1)The most effective concentrations of these two growth factors are 20 ng/ml bFGF and 200 ng/ml SDF-1.(2)The results demonstrated that ST2 cells treated with bFGF showed a significant increase in migration and proliferation.Compared with the SDF-1,the traditional growth factor used for migration,bFGF showed a stronger chemotaxis and proliferation promoting effect.(3)These changes were associated with increased activation of PI3K/AKT and Erk.(4)Inhibition of AKT with inhibitor LY294002 decreased cell viability and migration,and then bFGF could not activate cells growth anymore.Level of p-AKT was reduced significantly compared with control and bFGF-treated groups.Interestingly,we demonstrated that Erk signaling is responsible,at least in part,of the proliferation and migration of ST2 cells.Although Erk inhibitor U0126 could inhibit ST2 cell growth and wound-healing rate was significantly decreased,the activity of cells proliferation and migration could still be stimulated by bFGF even after U0126 was used,and p-Erk level was also increased in U0126+bFGF group.Conclusion:Collectively,these data demonstrate that PI3K/AKT and Erk pathway are both involved in ST2 cell proliferation and chemotaxis.In addition,bFGF facilitated ST2 cell proliferation and migration capacity mainly through PI3K/AKT pathway and partly through Erk pathway.