Effect of 1α,25-( OH)_2D_3 on the expression of RANKL and OPG mRNA in osteoblasts at different stage

Objective To investigate the effect of high-dose 1α,25-( OH)2D3on the changes of RANKL and OPG mRNA expression during osteoblast( OB) differentiation. Methods The osteogenic differentiation of MC3T3-E1 cells was induced in vitro. A 10nM concentration of 1α,25-( OH)2D3was additioned into the medium at the 0,7th,14th,21st,and the 28thday, respectively,in order to stimulate the osteogenic induction. Four hours after the stimulation,total RNA was extracted. And the expression of RANKL and OPG mRNA in OBs was detected using RT-PCR. Cells in control group were stimulated with absolute ethyl alcohol instead of 1α,25-( OH)2D3. Meanwhile,Alizarin red staining was carried out at the indicated different time-point,in order to detect the formation of mineralized nodules. Results The expression of RANKL mRNA in control groups showed no significant change,while the expression of OPG mRNA increased and reached the peak level at the 14thday,then decreased gradually. RANKL / OPG ratio was constanly lower than that at the base line( undifferentiated). 1α,25-( OH)2D3upregulated the expression of RANKL continuously,but it inhibited OPG gene expression. The RANKL / OPG ratio in study group was constantly higher than that in control group,especially during the mineralizing phase. And the difference was statistically significant( P 0. 05). Conclusion The regulating effect of 1α,25-( OH)2D3on the expression of RANKL and OPG changes at various differential stage of OBs.