不同浓度表儿茶素没食子酸酯对体外培养成骨细胞的影响

Effects of epigallocatechin gallate with different concentrations on osteoblasts cultured in vitro

目的研究不同浓度表儿茶素没食子酸酯(Epigallocatechin gallate,EGCG)对体外培养成骨细胞的影响,确定最大作用浓度(最大抑制浓度或促进浓度)。方法取新生SD大鼠的颅骨进行成骨细胞诱导培养,第3代细胞中分别加入浓度为0、1、10、20、30和40μmol/L的EGCG+含10%胎牛血清DMEM培养基培养。应用CCK-8法、茜素红染色和硝酸银染色法、ELISA法观察并测定EGCG对体外培养成骨细胞的增殖、矿化结节形成及血管内皮生长因子(VEGF)的影响。结果与0μmol/L浓度组比较,EGCG(10~20)μmol/L浓度组具有明显促进增殖的作用;30μmol/L及以上浓度组表现出对大鼠成骨细胞的抑制作用;在不同浓度EGCG中培养7 d或14 d,可以清晰观察到矿化组织形成,(10~20)μmol/L EGCG培养的细胞矿化结节形成数量显著多于对照组;血管内皮生长因子(VEGF)检测显示EGCG(10~20)μmol/L浓度组促进成骨细胞分泌VEGF的作用最强,30μmol/L及以上浓度对VEGF的分泌具有抑制作用。结论 EGCG(10~20)μmol/L浓度组对体外培养的成骨细胞有明显的促进增殖作用,30μmol/L及以上浓度组表现出对成骨细胞的抑制作用。

Objective To study the effects of different concentrations of epigallocatechin gallate（EGCG） on cultured osteoblasts in vitro and to determine the maximum concentration（maximum inhibitory concentration or promoting concentration）. Methods The skull of newborn SD rats was took to induce osteoblast. The cells of third generation were cultured in DMEM with 0, 1, 10, 20, 30 and 40 μmol/L EGCG ＋10% fetal bovine serum respectively. The effects of EGCG on proliferation, mineralized nodule formation and vascular endothelial growth factor（VEGF） in vitro were observed and determined by ELISA, CCK-8, alizarin red staining and silver nitrate staining methods. Results Compared with 0 μmol/L group, the concentration of（10-20）μmol/L EGCG could significantly promote the proliferation of rat osteoblasts at 30 μmol/L and above. When cultured in different concentrations of EGCG for 7 or 14 days, the number of mineralized nodules cultured in（10-20）μmol/L EGCG group was significantly more than that in control group. The group with 10-20 μmol/L EGCG promoted the most secretion of VEGF, groups with 30 μmol/L EGCG and above inhibited the secretion of VEGF. Conclusion At a concentration of（10-20）μmol/L, EGCG can significantly promote the proliferation of cultured osteoblasts in vitro. The inhibitory effects on osteoblasts were observed at concentrations of 30 μmol/L and above.