The Effects Of Sema3A On The Proliferation And Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells

Objective :1. To investigate the effects of Semaphorin3A(Sema3A) on the proliferation and osteogenic differentiation of BMSCs derived from Sprague Dawley rats. 2. To investigate the role of ERK1/2 signaling in Sema3A-mediated osteogenic differentiation of BMSCs.Methods :The isolated BMSCs were characterized by osteogenic and adipogenic differentiation and flow cytometry. 1. The cells were treated with Sema3 A at concentration of 0.5ug/ml or 1.0ug/ml. Cell proliferation was tested by MTT assay. The mRNA levels of ALP、osteocalcin( OCN)and Runt-related transcription factor2(Runx2) were investigated by quantitative real-time PCR(qRT-PCR) after 3 days culture. After 7 days, alkaline phosphotase(ALP) activity was detected using an ALP kit. Calcium deposition was assayed with Alizarin red staining(ARS) after 14 days culture. 2. The cells were treated with Sema3 A for 3 days, the protein levels of total ERK1/2 and phosphorylated ERK1/2(p-ERK1/2) was examined by western blot assay. BMSCs were treated with Sema3 A after that the ERK1/2 inhibitor PD98059 was added into cultures. Then the protein levels of total ERK1/2 and p-ERK1/2 was examined by western blot assay and the mRNA levels of ALP 、 OCN and Runx2 were investigated by qRT-PCR after 3 days culture. The mineralized nodules formation was assayed with ARS after 7 days culture.Results :BMSCs had been successfully induced osteogenic and adipogenic differentiation, and the result showed that cell surface antigen CD45(-), CD29(+), CD54(+) and CD90(+). 1. The proliferation of BMSCs treated with Sema3 A was not significantly different from control cells. qRT-PCR results showed that the genes expressions of ALP、OCN and Runx2 significantly declined in Sema3 A treated cells compared with controls(P<0.05). The ALP activity obviously decreased in Sema3 A treated cells(P<0.05). The difference was statistically significant. The ARS assay showed that the mineral nodule formation was also suppressed by Sema3 A. 2. Western blot assay showed that the expression of p-ERK1/2 was significantly decreased in osteogenic differentiation condition, Sema3 A could increased the level of p-ERK1/2. The protein level of p-ERK1/2 was decreased by ERK1/2 inhibitor PD98059.And the decrease of gene expression and mineralized nodule formation in Sema3 A treated cells was partly reversed by ERK1/2 inhibitor PD98059.Conclusion :These results suggest that Sema3 A had no obvious effect on the proliferation of BMSCs, but could inhibit osteogenic differentiation of BMSCs by ERK pathway.