Study On The Role Of Proton-sensing Receptor In Osteoblast Factor Expression And Its Molecular Mechanism

Bone metabolism is mediated by osteoblasts(Osteoblast)and osteoclasts(Osteoclast).Physiological or pathological conditions will bring about an acidic environment between cells or around cells,the acidic environment will promote osteoclast proliferation and improve its activity.RANKL is a key factor in the proliferation and activation of osteoclasts,which is expressed on the membrane of osteoblasts.Studies have shown that osteoblasts not only form the bone matrix in the process of bone formation,but also promote osteoclast differentiation.Some articles also prove that the acidic environment enhances the vitality of osteoclasts and inhibits the differentiation and mineralization of osteoblast,at the same time,it can activate the function of osteoblasts which is promoting osteoclast differentiation and maturation.The proton-sensing receptor GPCR can sense lipid molecules and protons,so the change of physiological activity of osteoblasts under acidic conditions has a certain relationship with GPCR.GPCR family contains OGR1,GPR4,TDAG8,G2A four receptors,it is a new research point of which one play the key role and the way it regulated under the acid conditions.At the same time,the study of its signal pathway not only provides a new theoretical support for the bone metabolism pathway in the basic theory,but also provides a new therapeutic target for osteoporosis and other bone diseases.Therefore,the study has a great significance in the theory and practice.In this experiment,the cell proliferation was measured by CCK method under acidic conditions,and the migration ability of hFOB cells was measured by Micro chemotaxis chamber method.We found that acidity on osteoblast proliferation and migration have a certain inhibitory effect.Collagen I?OPG?BMP2,the expression of the osteoblastic factors in human osteoblasts(hFOB)was detected by Real Time PCR.The expression of ALP was detected by ALP kit.The results show that acidic conditions inhibit osteoblast differentiation and ossification.Several important osteoclastic factors of TGF,RANKL and M-CSF were also detected by Real Time PCR.The results showed that the acidic condition had a certain effect on the expression of osteoclastic factors.TDAG8 and GPR4 were highly expressed in GPCR by qPCR detection of hFOB.We observed the expression of osteogenic factors and osteoclastic factors of hFOB after over-expression of two genes.We found that TDAG8 played a greater role in the whole regulation process,and GPR4 was used as an auxiliary effect.Similarly,the results of ELISA to determine PGE2 also involved in osteoblasts to promote the regulation of osteoclast differentiation in acidic conditions.Conclusion1.The ability of osteoblasts to form bone under acidic conditions is weakened,and the ability to promote osteoclast differentiation and maturation is improved.2.TDAG8 and GPR4 are involved in the regulation of physiological activities of osteoblasts under acidic conditions,in which TDAG8 plays a major role.TDAG8 can be used as a new drug target for the treatment of osteoporosis.3.TDAG8 and GPR4 are involved in the regulation of osteogenic factors and osteoclasts in osteoblasts.The expression of BMP2 and OPG decreased after transfection of TDAG8 and GPR4 respectively.The expression of OPG was more significant due to acidity,M-CSF expression is the same.The expression of RANKL is increased after over-expression of TDAG8 and GPR4,TDAG8 played a major role.While expression of TDAG8 increased the expression of TGF-?1.