Effects Of Phosvitin On Differentiation And Mature Of Osteoblast/Osteoclast Cells

Normal bone metabolism is a dynamic equilibrium process with the bone formation in osteoblast and bone resorption in osteoclast. When the balance disorders and bone resorption is greater than the bone formation, osteoporosis occurs. Recent studies found that phosphoproteins including osteopontin and bone sialoprotein, played a certain role in the regulation of bone metabolism process. Phosvitin is a highly phosphorylated protein which can affect the process of bone metabolism for its similar structure and composition with these proteins. In this paper, we study effects of phosvitin on the differentiation and mature of osteoblast and osteoclast cells and the corresponding mechanism on the cellular level, and the contents and results are as follows:(1) We systematically study effects of phosvitin on the MC3T3-E1 cellular proliferation, differentiation and mineralization and genetic mRNA expression of BMP-2 and Runx2 in osteoblast metabolism.In this paper, we adopt complete medium containing ascorbic acid(50μg/mL) and β-sodium glycerophosphate(100mmol/L) as osteoblast inducing conditional media and determine the proliferation and differentiation of phosvitin on the MC3T3-E1 cell by MTT method and ALP activity assay. The results show that phosvitin(1-1000μg/mL) has no significant impact on the growth of osteoblasts but can promote cell differentiation. And we observe effects of phosvitin on osteoblast mineralization through collagen Ⅰ content determination and alizarin-red staining, and find that phosvitin promote the formation of mineralized bone matrix maybe through promoting calcium deposition and synthesis, secretion of collagen Ⅰ. The results of real time fluorescent quantitative RT-PCR indicate that phosvitin could enhance BMP-2 and Runx2 genetic mRNA expression so as to promote bone formation of osteoblasts.(2) We study effects of phosvitin on the formation and bone resorption activity of osteoclast utilizing RAW264.7 preosteoclast cell models.In this paper, we adopt complete medium containing RANKL(50ng/mL) as osteoclast inducing conditional media and determine the proliferation of phosvitin on the RAW264.7 cell by MTT method firstly. And the results show that phosvitin in 1-500μg/mL concentration range has little influence on the growth of RAW264.7 cell. Then we operate tartrate resistant acid phosphatase staining and activity assay, and the results illustrate that the osteoclast inducing conditional media could induce osteoclast differentiation successfully and phosvitin(10μg/mL, 50μg/mL, 100μg/mL) could decrease osteoclast number and tartrate resistant acid phosphatase activity, thus inhabiting RAW264.7 cell differentiation in a dose-dependent manner. Real time fluorescent quantitative RT-PCR results show phosvitin can down-regulate expression of MMP-9, cathepsin K and RANK genetic mRNA and inhibit osteoclast activity of bone resorption.The dynamic balance between osteoblast and osteoclast makes normal bone metabolism. The effects of phosvitin can not only promote the regulation of bone formation in osteoblast, but also inhibit bone resorption in osteoclast. And all the data above may provide support and theoretical basis for phosvitin towards bone metabolism.