Effects Of PTH And Notch Signaling Pathway On The Differentiation Of Bone Mesenchymalstem Cells Into Osteoblasts

【Objective】: To study the effects of endogenous parathyroid hormone(PTH)and Notch signaling pathway on the differentiation of bone mesenchymal stem cells into osteoblasts, and to further explore the mechanism of its regulation.【Mathods】:In vivo selection of 8 weeks old PTH wild-type(WT, PTH+/+) and PTH gene knockout(KO, PTH-/-) male mice.To determine the role of PTH and Notch signaling pathway on the differentiation of bone mesenchymal stem cells into osteoblasts, the tibias were assessed by histology, immunohistochemistry.Bone mesenchymal stem cells obtained from mouse femurs were isolated and cultured,then osteogenic differentiation was induced. Western blot was performed to detect the expression of Notch signaling pathway at 3,7,10,14 days.The alkaline phosphate(ALP)activity was detected to evaluate the early osteogentic differentiation at 7 day.Western blot was performed to detect the expression of Runx2,OCN proteins.【Results】:Endogenous PTH deficiency inhibits the differentiation of bone marrow mesenchymal stem cells into osteoblasts, osteoblast number,Col I – positive area were reduced significantly in PTH gene knockout mice compared with WT. Notch1,Jagged1 immunohistochemical staining intensity was significantly reduced in PTH gene knockout mice.Western blot analysis showed the receptor and ligand of Notch signal pathway and the osteogenic index was increased in the wild type group and PTH gene knockout group after osteogenic differentiation.The expression of Jagged1 and Notch1 protein in PTH gene knockout group was significantly lower than the wild type group.Compared with the wild type group,Runx2 and OCN protein levels were lower in PTH gene knockout group. Mesenchymal stem cells induced osteoblasts, four days after the induction, Western blot detection test results showed that Notch1, Jagged1,Runx2 expression decreased, dbc AMP could correctable Notch1, Jagged1, Runx2 expression, and PKA inhibitor(H89) could eliminate the effect of dbc AMP. 7 days after the induction showed ALP activity decreased, giving dbc AMP correctable, plus PKA inhibitor(H89) to eliminate the effect of dbc AMP.【Conclusion】:The deficiency of endogenous PTH inhibits the differentiation of bone marrow mesenchymal stem cells into osteoblasts. This effect may be achieved by reduced the expression of Notch1 and Jagged1 proteins and mainly through the PKA/c AMP signaling pathway.