成纤维细胞生长因子-2对年轻和年老小鼠骨祖细胞增殖和分化的影响

Effects of fibroblast growth factor-2 on the proliferation and differentiation of osteoprogenitor cells from young and old mouse

目的：比较在不同浓度和培养时间下,成纤维细胞生长因子-2（fibroblast growth factor-2,FGF-2）对年轻和年老小鼠颅盖骨来源的骨祖细胞增殖的影响差异,并观察FGF-2对不同表型细胞的作用.方法：利用定时和连续的酶消化年轻（3个月龄）和年老（19～22个月龄）小鼠颅盖骨获取骨祖细胞,并进行原代培养.利用碱性磷酸酶（alkaline phosphatase,ALP）活性鉴定细胞的成骨活性.选取原代培养的骨祖细胞给予不同浓度的FGF-2,在不同的培养时间采用MTS法观察细胞增殖情况.采用两种成骨细胞谱系的分化标志物活化白细胞黏附分子（activated leukocyte cell adhesion molecule,ALCAM）和平滑肌肌动蛋白（smooth muscleactin,SMA）进行免疫荧光检测,观察FGF-2对不同表型细胞的作用.结果：质量浓度为1.6 ng/mL 和0.16 ng/mL的FGF-2,在培养4 h和24 h时对年轻小鼠骨祖细胞的增殖有促进作用,而对于年老小鼠的增殖促进作用出现在培养至48 h和72 h.年轻和年老小鼠的骨祖细胞中,均可表达ALCAM和SMA两种蛋白,FGF-2对SMA的阳性细胞比例没有明显影响,但可增加ALCAM阳性细胞的比例,并且这种促进作用对于年轻小鼠出现的更早.结论：适宜质量浓度的FGF-2可以促进小鼠骨祖细胞的增殖,与年轻小鼠相比,年老小鼠的骨祖细胞对FGF-2的反应性下降.FGF-2可能对促进老年骨增量中具有潜在治疗价值.

Objective： To compare the different effects of fibroblast growth factor（FGF-2） on both proliferation and differentiation of osteoprogenitor cells from young and old mouse ealvaria with different concentrations and cultured time and to observe which phenotype of the cells are affected by FGF-2 through immunofluorescenee staining. Methods： Calveria from young and old mice were enzymatically digested and fraetionated to obtain osteoprogenitor cells. To prove the cells used in our experiment are osteoprogenitor cells which have osteogenetie activity, the activity of alkaline phosphatase（ALP） were tested. MTS assays were used to observe the rate of cell proliferation under different concentrations of FGF-2 and cultured time. Immunoeytoehemistry was performed for activated leukocyte cell adhesion molecule（ALCAM） and smooth muscleactin（SMA） of the osteoblast lineage to determine the phenotype of the cells that were stimulated to proliferate by FGF-2. Results： The con- centration 1.6 ng/mL and 0.16 ng/mL of FGF-2 enhance the proliferation of the osteoprogenitor cells from both young and old mouse compared to control group. FGF-2 enhance the proliferation of the osteoprogenitor cells from young mouse up to 4 h and 24 h, but for old mouse, the effect appeared until 48 h and 72 h. There was no significant effect of FGF-2 （0.16 ng/mL） on the percentage of SMA positive cells, but FGF-2 could significant increase the percentage of ALCAM positive cells in young mouse up to 24 h（P〈0.05） and for old mouse, the effect appeared until 72 h. Conclusion： The appropriate concentration of FGF-2（0.16 ng/mL, 1.6 ng/mL） could stimulate the proliferation of mouse osteoprogenitor cells. Compared to young mouse, the responsiveness of the osteoprogenitor cells from old mouse decreased, but the effect of different concentrations of FGF-2 on the osteoprogenitor cells from young and old mouse had no difference.