1 Alpha, 25 (oh) < Sub > 2 < / Sub > D < Sub > 3 < / Sub > Effect On Osteoclast Formation And Vitality

Bone tissue is continuously updated and maintaining vitality, which depends on bone metabolic balance. Disruption of this balance will lead to metabolic bone disease. Animal bone malnutritions main attribute to the fact that, bone resorption caused by osteoclasts (OC) is more serious than bone formation caused by osteoblasts (OB). OC formation and activation are mainly controlled by Receptor Activator of NF-κB Ligand (RANKL) expressed in osteoblasts. Present studies suggest that,1α,25-(OH)2D3can regulate bone metabolism not only by promoting calcium and phosphorus absorption of intestinal mucosa or calcium and phosphorus reabsorption of renal, but also by acting directly on osteoblasts through RANKL/RANK/OPG system. There were also few researchs showed that,1α,25-(OH)2D3can act directly on OC. Therefore, further understanding of the precise regulating mechanisms of vitamin D on bone cells is important for prevention and cure of metabolic bone disease.In this paper, osteoclast precursor cells RAW264.7were used to explore the effects of different culture methods on the characteristics of cell biology. And on this basis, we studied the effects of different concentrations of RANKL (25、50、100ng/ml)、1α,25-(OH)2D3(1×10-9、1×10-8、1×10-7mol/L) on OC proliferation, differentiation and bone resorption. The results showed that:(1) Cells would be digested after10～15min treated by trypsin one time, meanwhile two times digestion needed only3-5minutes. Shorter time digestion reduced cell injury. So, they had better adherent ability and good shape as well as elevated proliferation rate. Cells survival rate after resuscitation under slow-freezing method (88.7%) was higher than those under rapid-freezing method (81.8%) very significantly (P<0.01).These results demonstrated that two times trypsin digestion and slow-freezing method were beneficial for RAW264.7growth and reproduction.(2) After three days culture, different concentrations of RANKL inhibited cell proliferation dose dependently. There had very significantly differences between the control group and the50or100ng/ml RANKL group (P<0.01). Meanwhile, the number of TRAP-positive polynuclear cells in the50or100ng/ml RANKL group was very significantly higher than that in the control group or25ng/ml RANKL group (P<0.01). There were some obvious absorption lacunae on the surface of ivory discs in each RANKL group. However, compared with the control group, different concentrations of1α,25-(OH)2D3promoted cell proliferation dose dependently after three days culture. And there were more serious absorption lacuna as well as more TRAP-positive polynuclear cells in the group containing1α,25-(OH)2D3than those in the group containing RANKL alone. These results demonstrated that RANKL could inhibit cell proliferation and promote the formation and activation of OC, meanwhile1α,25-(OH)2D3could promote cell proliferation and enhanced the formation and absorption of OC induced by RANKL.