| Bone is a dynamic organ. Osteoblast’s (OB) bone formation and osteoclast’s (OC) bone resorption may dominate at a given point, or bone metabolic diseases, such as osteoporosis and osteoaclerosis may happen, 1α,25-(OH)2D3 have the strongest bioactivity metabolites of Vitamin D, as a steroid hormone, which can regulate the calcium and phosphorus metabolism and plays an important role in maintaining calcium homeostasis. TRPV5, found in the kindney tubule, shows a high sensitivity to Ca2+. Studies have been found that lacking of TRPV5 channel will affect the differentiation and bone resorption of osteoclasts, but the associated mechanism remains unclear. This research has mainly been focused on the effects of TRPV5 in the differentitaion and resorption of OC under regulation of Vitamin D. To provide a theoretical basis for the further clarify of bone resorption activity of OC-related regulatory mechanism, a series of tests were carried out:1.The effects of seeding density on the differentiation of osteoclasts under the regulation of Vitamin DThis study aimed to investigate the effects of seeding concentration on the differentiation of osteoclasts.The bone marrow mononuclear cells are isolated from 4-5week old BALB/c mices’ bone marrow and seeded in the 96-well plates with concentrations of 0.3,0.7,1.5,3,6,12,24, 48×104/well (0.94,2.18,4.68,9.37,18.75,37.5,75,150 X 104/cm2). The bone marrow mononuclear cells are incubated with 30 nmol/L M-CSF and 50 nmol/L RANKL for 2 d in presence or absence of 10 nmol/L 1,25(OH)2D3 before TRAP stain, followed by an additional 3d incubation in the presence of 50 nmol/L M-CSF and 30 nmol/L RANKL with serum-contained a-medium. The immunofluorescence was used to detect the expression level of VDR in osteoclast. Results show that osteoclasts formation increased with the increasing concentration of mononuclear cells. Osteoclast generation rate was negatively correlated with cells seeding density, the higher the seeding density, the lower generation rate of osteoclast. VDR distributed in mononuclear cells, immuature osteoclasts and muature osteoclast. The effects of Vitamin D on differentiation of OC depend on the seeding density. Vitamin D significantly inhibit the group seeded in 18.75,37.75,75 X 104/cm2 concentration differentitaion compared with the control group. While in the density of 0.93 and 2.18×104/cm2, the differentitation of OC was increased by Vitamin D.2. The effects of TRPV5 on the differentitaion and resorption of OCTo study the effects of TRPV5 on the differentitaion and resorption of OC, RuR was used as the blocker of TRPV5 to research the effects of different concentrations (0,10,50,100,500 and 1000 nmol/L) of RuR on osteoclast differentiation. The western-blot was used to test the level of TRPV5 expressed in different concentrations of RuR at different times (1d,2d,3d). Meanwhile, the osteoclastic bone resorption markers protein like, V-ATPase, CTSK, TRAP and Call were also examined. The results show that:0,10,50,100,500 and 1000 nmol/L RuR didn’t have effects on osteoclast activity, but the ability of osteoclastogenesis was inhibited. The expression level of TRPV5 in osteoclast was inhibited by different concentrations (10,100, 1000nmol/L) of RuR at different times (12h,24h,72h). Furthermore, the expression level of the osteoclastic bone resorption markers protein like, V-ATPase, CTSK, TRAP and Call decreased to a certain extent, as well as the NFATc1.3. The effects of TRPV5 on the differentitaion of OC under the regulation of Vitamin DTo characterize the role of TRPV5 in Vitamin D-regulated osteoclastogenesis, the osteoclast are cultured with different concentrations of Vitamin D under different times. Meanwhile, The osteoclast was treated with Vitamin D, Vitamin D+TRPV5 blocker (RuR/Eco) and TRPV5 blocker (RuR/Eco), at 2 d,4 d,6 d for two days, respectively, and TRAP-staining was used to study the role of TRPV5 in Vitamin D-regulated osteoclastogenesis. Meanwhile, the expression level of TRPV5, the resorption markers protein like, V-ATPase, CTSK, TRAP and Call, osteoclast transcription factor NFATc1 were detected by Western blot. The results show that, Vitamin D inhibited the TRPV5 expression only in 1 d and 2 d. The inhibition of osteoclastogenesis by Vitamin D only at 2 d, but with Vitamin D+TRPV5 blocker (RuR/Eco) the inhibition were at all time, while there is no significant difference between Vitamin D group and TRPV5 blocker (RuR/Eco). Compared with Vitamin D group, Vitamin D+TRPV5 blocker (RuR/Eco) significantly inhibited the expression level of the resorption markers protein like, V-ATPase, CTSK, TRAP and Call, while no significantly influence on NFATc1. The above results show that, Vitamin D inhibit the osteoclastogenesis from mononuclear cells through reduceing the expression level of TRPV5 therefore inhibit TRPV5-mediated activation of NFATc1 by RANKL. |
