摘要目的 观察氟通过胞外信号调控激酶(ERK)通路对成骨细胞(MC3T3-E1)增殖作用的影响.方法 取小鼠成骨细胞(MC3T3-E1)进行体外培养,加入不同浓度的氟(Fˉ,0、200、400、600、1000、2000、4000、8000、10 000 μmol/L)培养24、48 h,甲基噻唑蓝(MTT)法筛选出促进细胞增殖的最适浓度.根据最适浓度,将成骨细胞单纯随机分为3组:对照组(Fˉ,0 μmol/L)、染氟组(Fˉ,400 μmol/L)、染氟抑制组(Fˉ,400 μmol/L+PD9805,10 μmmol/L).培养48 h后应用流式细胞术检测各组细胞周期;Western bolt法和免疫荧光法检测各组磷酸化ERK(P-ERK)蛋白表达.结果 400 μmol/L的氟是促进成骨细胞增殖的最适合浓度.与对照组比较[(76.12±10.08)％、(2.06±0.31)％],染氟组G0/G1期细胞数[(63.04±8.12)％]明显减少,S期细胞数[(9.13±2.08)％]明显增多(P均＜ 0.05);而染氟抑制组G0/G1期细胞数[(92.11±9.01)％]明显增多(P＜0.05).Western blot结果表明,与对照组[(100.00±0.00)％]比较,染氟组成骨细胞P-ERK蛋白表达水平[(131.24±13.88)％]明显增高(P＜0.05),染氟抑制组P-ERK蛋白表达[(91.33±9.68)％]未见明显改变(P＞0.05);免疫荧光法检测结果与Western blot法相似.结论 400 μmol/L氟可以促进成骨细胞增殖,ERK通路在氟促进成骨细胞的增殖作用中起到了关键的作用.

abstractsObjective To study the effect of fluorine on proliferation of osteoblast through extra cellular signal-regulated protein kinase(ERK) signaling pathway.Methods Mouse osteoblasts(MC3T3-E1) were cultured in vitro with different concentrations of fluoride for 24 and 48 h (the concentrations of Fˉ were 0,200,400,600,1000,2000,4000,8000,10 000 μmol/L,respectively).The optimum concentration for promotion of cell proliferation was determined by methylthiophene tetrazolium(MTT) assay.According to the optimum concentration,the cells were randomly divided into three groups:control group (0 μmol/L Fˉ); fluorine group (400 μmol/L Fˉ); fluorine and MAPK inhibitor PD98059 group(400 μ mol/L Fˉ + 10 μ mmol/L PD98059).Cell cycle was detected by flow cytometry after 48 h culture.The expression of P-ERK protein was determined by Western blotting and immunofluorescence.Results The optimum concentration of fluorine for proliferation of osteoblasts was 400 μ mol/L.Compared with the control group[(76.12 ± 10.08)％,(2.06 ± 0.31)％],the number of cells in G0/G1 phase[(63.04 ± 8.12)％] reduced and the number of cells in S phase[(9.13 ± 2.08)％] increased in fluorine group (all P ＜ 0.05) ; but the number of cells in G0/G1 phase [(92.11 ± 9.01 ) ％] in fluorine and mitogen-activated protein kinases (MAPK) inhibitor PD98059 group was significantly increased(P ＜ 0.05 ).Western blotting results showed that:compared with the control group[(100.00 ± 0.00)％],the expression of P-ERK protein in fluorine group[(131.24 ± 13.88)％] was significantly higher(P ＜ 0.05 ),but the expression of P-ERK protein in fluorine and MAPK inhibitor PD98059 group [(91.33 ± 9.68 )％] was not significantly changed(P ＞ 0.05).The results of immunofluorescence were similar to that of Western blotting.Conclusions Fluorine at the concentration of 400 μmol/L can promote the proliferation of osteoblasts.ERK signaling pathway has played a key role in the proliferation of osteoblasts.