1,25-二羟基维生素D_3对小鼠骨髓细胞形成破骨细胞样细胞及其骨吸收效应的影响

Effects of 1,25-dihydroxyvitamin D_ 3 on stimulation of osteoclast-like cell formation and bone resorption in primary murine marrow cell culture

目的 动态观察 1,2 5-二羟基维生素 D3[1,2 5- ( OH) 2 D3]对 NH小鼠骨髓细胞培养在体外形成破骨细胞及其骨吸收的剂量效应和作用时象。方法 收集 NH小鼠骨髓细胞于含 10 %胎牛血清的α- MEM培养基中行体外培养 ,设置不同的 1,2 5- ( OH) 2 D3浓度组和给药时间组 ,并于培养第 3、6、9、12天观察记录抗酒石酸酸性磷酸酶 ( tartrate- resistant acid phosphatase,TRAP)阳性多核巨细胞 [或破骨细胞样细胞 ( osteroblast- like cell,OL C) ]以及骨磨片上骨吸收陷窝的数目。结果 高于 10 -9mmol/L的1,2 5- ( OH) 2 D3单一刺激可于培养第 6天诱导 TRAP阳性 OLC形成 ,并且可在骨磨片上观察到骨吸收陷窝 ;随 1,2 5- ( OH) 2 D3浓度的增高 ,OLC和骨吸收陷窝数目均随之增加 ;各浓度组 OLC数目于培养第9天达最高值 ,随后则趋于减少 ,而骨吸收陷窝数于培养第 9和第 12天均有所增加 ;采用单一 1,2 5-( OH) 2 D3浓度 ( 10 -8mmol/L) ,TRAP阳性 OL C和骨吸收陷窝见于培养 3d后用药和全程用药组 ,而仅在培养前 3d用药则不能诱导 OL C和骨吸收陷窝的形成。结论 1,2 5- ( OH) 2 D3可在体外诱导骨髓单核细胞分化形成 OLC并使其具有破骨活性 ,诱导作用的强弱与 1,2 5- ( OH) 2 D3的浓度相关 ,而且其作用时象可能是在培?

Objective To clarify the events involved in 1,25 (OH) 2D 3\|induced osteoclast\|like cell (OLC) formation and bone resorption,we have characterized the concentration effect and the timing of 1,25 (OH) 2D 3 stimulation in primary murine marrow cell culture. Methods\ Marrow cells from NH mouse were harvested and cultured in α MEM with 10% fetal bovine serum.The appearance of tartrate\|resistant acid phosphatase (TRAP)\|staining multinuclear cells (or OLCs) and formation of bone resorption pits were measured after 3,6,9 and 12 day exposure to various concentrations of 1,25\|(OH) 2D 3 in culture. Results\ 1,25 (OH) 2D 3 (concentration higher than 10 -9 mol/L) could induce recruitment of OLCs and their bone resorption activity in a dose dependent manner.Formation of TRAP+ve OLCs was first observed on day 6,reached to its peak on day 9 and reduced in number after the day 9 time point.The number of bone resorption pits on the bone slices in culture showed a consistent increase on day 9 and day 12.In cultures to which 1,25\|(OH) 2D 3 (10 -8 mol/L) was added only for the first 3 days,TRAP+ve OLCs and bone resorption pits were not present on day 6,whereas continuous treatment with 1,25\|(OH) 2D 3 starting from the beginning or from day 3 of the experiment invariably stimulated OLC formation and bone resorption activity. Conclusions\ 1,25\|(OH) 2D 3 can induce OLC formation and bone resorption in murine marrow cell culture.The induction effect is dose dependent and is likely to occur after 3 days of culture.