| Osteoclast (OC) are the unique bone resorption cell in vivo, and its brisk of proliferation and differentiation or hyperfunction of activity could cause a variety of bone diseases. Differentiation and function of OC are effected by many factors, such as receptor activator of nuclear factor-κB ligand (RANKL), receptor activator of nuclear factor-Kb (RANK), osteoprotegerin (OPG), etc. Studies demonstrated that the "RANKL/RANK/OPG" system is an important signaling pathway during OC differentiated process, in which RANKL and OPG secreted by osteoblast (OB) regulate OC differentiation through combining with RANK on the surface of osteoclast precursor cell (OPC) and OC competitively. Researches show that1alpha,25-dihydroxyvitamin D3(1α,25-(OH)2D3), the major active form of vitamin D, could adjust the the expression of RANKL and OPG in OB to regulate bone metabolism indirectly. In addition, a large number of studies indicated that OPC and OC could express the vitamin D receptor. However, the mechanism of OC formation and activation regulated by vitamin D directly is still not clear. To clear the part mechanism on the regulation of OCs’ formation and differentiation by vitamin D, RAW264.7cells as OPC, were induced to be OC by macrophage colony stimulating factor (M-CSF) and RANKL in this study. On the base, different concentrations of la,25-(OH)2D3(10-7,10-8and10-9mol/L) were added into the culture medium. Then, the formation, bone resorption activity, the expression of OC functional protein (integrin αvβ3, V-ATPase, CAII, CTSK, TRAP and MMP-9) and key transcription factors (c-Fos, NFATcl) were detected. It designes to indicate the partial mechanism about differentiation and activation of OC which regulated by vitamin D.1. Establishment on the culture mode of OC induced from RAW264.7cellsDuring RAW264.7cells culture,25μg/L M-CSF and0,10,30,50or100μg/L RANKL were added into the culture medium in vitro. The rate of cells proliferation was measured by MTT assay, the formation and differentiation of osteoclasts were confirmed via tartrate-resistant acid phosphatase (TRAP) staining and bone absorption analysis. The results showed that, M-CSF plus different concentrations of RANKL could suppress the proliferation of RAW264.7cells and promote the formation and activation of OC in dose-dependent. Among them, a large number of OC could be induced by25μg/L M-CSF combined with30μg/L RANKL to meet molecular biology research demand.2. Effects on the formation and bone resorption activity of OC treated by1α,25-(OH)2D3The aim of this study was to investigate the effects on the formation and activation of OC treated by1α,25-(OH)2D3. RAW264.7cells were cultured by different concentrations of la,25-(OH)2D3(0,10-7,10-8and10-9mol/L)and ethanol solvent control, while they were induced by25μg/L M-CSF and30μg/L RANKL in vitro. The rate of RAW264.7cells proliferation was measured by MTT assay, and the formation and differentiation of OC were confirmed via tartrate-resistant acid phosphatase (TRAP) staining counted and bone absorption analysis. The results showed that, different concentrations of1α,25-Dihydroxyvitamin D3inhibited the proliferation of RAW264.7cells and promoted the formation and bone resorption activity of OC, while ethanol solvent had no significant effect on Inconclusion, the formation and activation of OC could induced by1α,25-(OH)2D3.3. Effects on the expression of OC functional protien treated by1α,25-(OH)2D3The aim of this study was to investigate the effects on the expression of OC functional protein, including integrin αvβ3, V-ATPase, CAII, CTSK, TRAP and MMP-9during its formation and activation treated by1α,25-(OH)2D3. RAW264.7cells were treated by different concentrations of1α,25-(OH)2D3(0,10-7,10-8and10-9mol/L), while they were cultured with25μg/L M-CSF and30μg/L RANKL in vitro. The expression of functional protein was detected by western blot. The results showed that, the expression of integrin αvβ3, V-ATPase, CAII, CTSK, TRAP and MMP-9was enhanced after treating by1α,25-(OH)2D3on early stage (24,48h).10-8mol/L1α,25-(OH)2D3had the most obvious effect. With the extension of incubation time (72h), the expression of integrin αvβ3, V-ATPase, CAII, CTSK, TRAP and MMP-9was down-regulated after treating by1α,25-(OH)2D3. Inconclusion, the expression of OC functional protein was accelerated by1α,25-(OH)2D3during its formation and activation.4. Effects on the expression of OC key transcription factors treated by1α,25-(OH)2D3The aim of this study was to investigate the effects on the expression of OC key transcription factors, c-Fos and NFATcl, by1α,25-(OH)2D3during its formation and activation. RAW264.7cells were treated by different concentrations of1α,25-(OH)2D3(0,10-7,10-8and10-9mol/L), while they were cultured with25μg/L M-CSF and30μg/L RANKL in vitro. The expression of OC key transcription factors was detected by Q-RT-PCR. The results showed that, the expression of c-Fos and NFATcl was increased after treating by10-8mol/L1α,25-(OH)2D3on early stage (24h). With the extension of incubation time (48,72h), the expression of c-Fos and NFATcl was down-regulated after treating by1α,25-(OH)2D3. In conclusion, the expression of OC key transcription factors, c-Fos and NFATc1, was promoted by1α,25-(OH)2D3during its formation and activation. |
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