PDGF-BB Promotes Proliferation And Differentiation Of MC3T3-E1 Cells Through SRC-JAK2 Signaling Pathway

Objectives:This experiment is aimed to detect whether Src,JAK2 was expressed in MC3T3-E1 cells and whether Src lay in the upstream of JAK2 in MC3T3-E1 cells,and whether PDGF-BB could activate Src-JAK2 signaling pathway,so that to further investigate whether PDGF-BB have an effect on differentiation and proliferation of MC3T3-E1 cells through Src-JAK2 signaling pathway.Methods:Firstly,the expression of Src,JAK2 in MC3T3-E1 cells and the effect of PDGF-BB on them were inspected by means of Western blot.Later,Western blot was also used to investigate whether Src lay in the upstream of JAK2.Afterwards the influence of PDGF-BB on MC3T3-E1 cells poliferation was detected by CCK-8,and the inhibitor of PDGFR?,AG1295 and the inhibitor of Src,SU6656 and the inhibitor of JAK2,AG490,were used to examine the role of Src-JAK2 signaling pathway in MC3T3-E1 proliferation induced by PDGF-BB.Then,Real-time PCR and Western blot were used to test whether PDGF-BB could promote MC3T3-E1 cells osteogenic differentiation.Similarly,the inhibitor of PDGFR?,AG1295 and the inhibitor of Src,SU6656 and the inhibitor of JAK2,AG490,were used to examine the role of Src--JAK2 signaling pathway in MC3T3-E1 osteogenic differentiation induced by PDGF-BB.Finally,MC3 T3-E1 cells osteogenic differentiation and mineralization were examined by ALP stain and alizarin stain.The same method was used to determine whether Src-JAK2 signaling pathway play a role in MC3T3-E1 cells osteogenic differentiation and mineralization.Results:Src,JAK2 were expressed in MC3T3-E1 cells and PDGF-BB could activate Src,JAK2 in a time dependant manner within 30 minutes.Src lay in the upstream of JAK2 in MC3T3-E1 cells.Therefore,we proved that PDGF-BB have the ability to activate Src-JAK2 signaling pathway in MC3T3-E1 cells.PDGF-BB was able to promote MC3T3-E1 cells proliferation and this effect was suppressed by AG1295,SU6656,AG490.Similarly,we found that PDGF-BB could promote the expression of osteogenic relative gene such as,Runx2,Osterix,OCN,OPN,Collal,and relative protein such as,Runx2,Col1?1.What's more,we also discovered that PDGF-BB was able to induce osteoblasts mineralization.Osteogenic differentiation and mineralization of MC3T3-E1 cells promoted by PDGF-BB were suppressed by AG1295,SU6656 and AG490.Conclusions:PDGF-BB could promote MC3T3-E1 cells proliferation and differentiation through Src-JAK2 signaling pathway.