1α，25(OH)2D3对不同分化阶段成骨细胞RANKL及OPG基因表达的影响

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

目的研究高剂量1α,25(OH)2D3对RANKL/OPG表达的调控是否随成骨细胞(osteoblasts,OB)分化成熟而变化。方法体外诱导MC3T3-E1细胞成骨分化,并分别于成骨诱导第0、7、14、21、28 d向细胞加以1α,25(OH)2D3(10 nM)的刺激,4 h后提取细胞总RNA,进行RT-PCR检测OB RANKL及OPG mRNA表达水平。对照组则以无水乙醇刺激。同时,对不同分化阶段OB进行茜素红染色,以检测钙结节的形成。结果在OB分化过程中,对照组RANKL表达无显著变化;OPG基因表达逐渐增多,在第14天达最高水平,之后逐渐下降;RANKL/OPG比值始终低于未成骨诱导时的RANKL/OPG比值。1α,25(OH)2D3持续促进RANKL基因的表达,并且持续抑制OPG基因表达;实验组RANKL/OPG比值一直高于对照组,尤其在矿化阶段,差异有统计学意义。结论 1α,25(OH)2D3对RANKL和OPG的调控随OB分化成熟而变化。

Objective To investigate the effect of high-dose 1 α,25-（ OH）2D3 on 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） 2D3 was additioned into the medium at the 0, 7th, 14th, 21st, and the 28th day, 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 I a ,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 14th day, then decreased gradually. RANKL/OPG ratio was constanly lower than that at the base line （ undifferentiated）. 1 α,25-（OH） 2D3 upregulated 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）2D3 on the expression of RANKL and OPG changes at various differential stage of OBs.