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目的研究不同浓度1,25二羟维生素D3[1,25(OH)2D3]对小鼠成骨细胞增殖分化及维生素D受体(VDR)表达的影响,探讨VDR在成骨细胞增殖分化中的作用。方法在成骨细胞株MC3T3E1培养液中加入不同浓度的1,25(OH)2D3(10-8、10-9、10-10mol/L),培养48h后应用噻唑蓝(MTT)比色法检测其对成骨细胞增殖的影响,采用细胞培养上清中骨钙素(OC)水平分析方法检测1,25(OH)2D3对成骨细胞分化的影响;采用SYBR Green荧光定量PCR方法检测细胞VDR基因mRNA表达,Western blot法检测细胞VDR蛋白水平表达。结果1.小鼠成骨细胞在1,25(OH)2D3处理48h后,各实验组(10-8、10-9、10-10mol/L组)吸光度均低于对照组,差异均有统计学意义(Pa<0.05);2.1,25(OH)2D3处理后,10-8、10-9mol/L组细胞上清中OC水平均高于对照组(Pa<0.05),而10-10mol/L组OC水平及VDR基因mRNA表达量与对照组比较差异均无统计学意义(Pa>0.05);3.荧光定量PCR及Westernblot结果显示10-8、10-9mol/L处理组细胞VDR基因mRNA及蛋白表达均显著高于对照组(Pa<0.05),10-10mol/L组与对照组比较无显著性差异(P>0.05)。结论1,25(OH)2D3可显著抑制成骨细胞增殖、促进成骨细胞分化,且可促进成骨细胞VDR表达,提示VDR信号通路在成骨细胞的增殖分化中可能起一定作用。
Objective To investigate the effects of different concentrations of 1,25-dihydroxyvitamin D3 [1,25 (OH) 2D3] on the proliferation and differentiation of osteoblast and the expression of vitamin D receptor (VDR) in mice and to explore the role of VDR in osteoblast proliferation and differentiation effect. Methods Different concentrations of 1,25 (OH) 2D3 (10-8, 10-9, 10-10mol / L) were added to the culture of osteoblasts MC3T3E1 and cultured for 48 hours. MTT colorimetric assay The effect of 1,25 (OH) 2D3 on osteoblast differentiation was examined by using osteocalcin (OC) level analysis method in cell culture supernatant. SYBR Green fluorescence quantitative PCR was used to detect the cell VDR The mRNA expression of VDR was detected by Western blot. After treated with 1,25 (OH) 2D3 for 48h, the absorbance of all the experimental groups (10-8,10-9,10-10mol / L) were lower than that of the control group (P0.05). After treated with 2.1,25 (OH) 2D3, OC levels in supernatant of 10-8,10-9 mol / L group were higher than those in control group (Pa <0.05) There was no significant difference in OC level and VDR gene mRNA expression between L group and control group (P> 0.05) .3. Fluorescent quantitative PCR and Western blot showed that VDR gene mRNA expression in 10-8,10-9mol / L group And protein expression were significantly higher than the control group (Pa <0.05), 10-10mol / L group and the control group no significant difference (P> 0.05). Conclusions 1,25 (OH) 2D3 can significantly inhibit the proliferation of osteoblasts, promote the differentiation of osteoblasts and promote the expression of VDR in osteoblasts, suggesting that VDR signaling may play a role in the proliferation and differentiation of osteoblasts.