Role Of Neuropeptide Substance P And BMP Signaling Pathway In Osteogenic Differentiation Of ST2 Cells

Part one:Effects of Substance P on the proliferation and differentiation of ST2 cellsObjectives:To investigate the effect of Neuropeptide Substance P(SP)on the proliferation and differentiation of ST2 cells during osteogenic differentiation,and to screen out the appropriate SP concentration and time for promoting proliferation and differentiation.Methods:ST2 cells of the third passage were inoculated into a 96-well culture plate at a concentration of 5×103/well,the cells were stimulated with osteoblast medium at different concentrations of SP(10-10Mol/L,10-8Mol/L,10-6Mol/L,10-5Mol/L),that contains 2%FBS alone was used as a control.Each sets of five holes and a zero-hole adjustment.The cells were stimulated with SP for 24、48、72 hours respectively,and the proliferation rate was detected by CCK-8.To screen out the appropriate concentration.Cells of the third generation were seeded in a 6-well cell culture plate at 8×104 cells per well.Complete medium containing the 10-6Mol/L SP(containing 2%FBS)was added and cultured for 1,3,5 and 7 days.The expression of alkaline phosphatase(ALP),Collagen typy-Ⅰ(Colla Ⅰ),osteocalcinv(OCN)in the culture supernatant was tested by ELISA.And the activity of ALP was detected by immunofluorescence staining.Results:(1)Neuropeptide substance SP could promote the proliferation of mouse bone marrow mesenchymal stem cells(ST2 cells),that had certain correlation with SP concentration and time.When SP concentration was 10-6Mol/L,the effect of cell proliferation was the most obvious.(2)Osteoblast differentiation of mouse bone marrow mesenchymal stem cells(ST2 cells)was promoted by neuropeptide substance P(SP).The expression of early differentiation marker ALP reached its peak at day 5,and the late differentiation marker Colla Ⅰ and OCN peaked at day 7.SP can enhance the ALP activity of ST2 cells.Conclusions:Neuropeptide P substance(SP)promotes the proliferation and osteogenic differentia-tion of mouse BMSCs(ST2 cells)in a dose-dependent and time-dependent manner.Part two:Regulation of BMP signaling pathway on the osteogenic differentiation of SP promoting ST2 cells.Objectives:To investigate the role of BMP signaling pathway in the osteogenic differentiation of SP promoting ST2 cells.Methods:Cells of the third generation were seeded in a 6-well cell culture plate at 8×104 cells per well,and every group sites three holes.Experimental Packet Processing:SP group:10-6Mol/L SP;SP+Noggin group:10-6MOl/L SP and 100ng/ml Noggin;Noggin group:100ng/ml Noggin;control group:equivalent 2%FBS.The expression of ALP,ColIa Ⅰ and OCN in the culture supernatant of cells was detected by ELISA after 5 or 7 days.The the activity of ALP was detected by immunofluorescence staining.Results:(1)SP can significantly promote the expression of osteogenic differentiation markers ALP,ColIal and OCN in ST2 cells.After adding Noggin,the BMP signaling pathway inhibitor,the expression of osteogenic differentiation markers decreased significantly,and the addition of Noggin alone also inhibited ALP,ColIaI and OCN expression.(2)Immunofluorescence staining showed that SP could promote the expression of ALP in ST2 cells.After adding Noggin,the activity of ALP was inhibited.The addition of Noggin alone could also inhibit the expression of ALP.Conclusions:SP can promote the osteogenic differentiation and ALP activity expression of ST2 cells,and Noggin can inhibit the role of play.It suggests that BMP signaling pathway may play a regulatory role in the process of SP promoting ST2 osteogenic differentiation.