目的探讨氟对体外培养大鼠成骨细胞中骨保护素(OPG)/核因子-κB受体活化因子配体(RANKL)/核因子一KB受体活化因子(RANK)信号传导通路的影响。方法取新生24h内SD大鼠仔鼠,应用酶消化法分离大鼠颅骨成骨细胞,取第2代成骨细胞,加入不同浓度[O(对照)、1×10^-3、1×10^-4、1×10^-5、1×10^-6、1×10^-7mol/L]的氟,培养24、48h后,采用实时荧光定量PCR法检测成骨细胞RANKL、OPGmRNA表达;蛋白免疫印迹(Westernblot)法检测成骨细胞RANKL、OPG蛋白表达。结果①染氟24h,成骨细胞RANKL、OPGmRNA表达组间比较差异有统计学意义(F值分别为30.95、22.62,P均〈0.01),其中1×10^-5mol/L组成骨细胞RANKL mRNA(5.99±0.39)和1×10^-4、1×10^-5、1×10^-6mol/L组OPG mRNA(3.52±0.09、4.81±0.15、3.68±0.04)的表达高于对照组(3.20±0.19、3.09±0.58,P均〈0.05),1×10^-3mol/L组RANKL mRNA(2.29±0.18)的表达低于对照组(P均〈0.05);染氟48h,成骨细胞RANKL、OPG mRNA的表达组间比较差异有统计学意义(F值分别为26.62、5.72,P均〈0.01),其中1×10^-5mol/L组成骨细胞RANKL、OPGmRNA(6.67±0.49、5.05±0.51)的表达高于对照组(4.29±0.07、4.34±0.12,P均〈0.05),1×10^-3 mol/L组OPG mRNA(3.63±0.49)的表达低于对照组(P〈0.05)。②成骨细胞RANKL蛋白表达在染氟24、48h后,组间比较差异无统计学意义(F值分别为0.07、0.49,P均〉0.05)。成骨细胞OPG蛋白的表达,染氟24h后组间比较差异有统计学意义(F=3.26,P〈0.05),1×10^-5mol/L组(1.45±0.10)高于对照组(1.05±0.06,P〈0.05);染氟48h,成骨细胞OPG蛋白表达组间比较差异无统计学意义(F=0.44,P〉0.05)。结论氟在低浓度时以成骨活动为主;但随着染氟时间和浓度的增加,促进破骨细胞的分化、?
Objective To study the influence of fluorine on signaling pathway of osteoprotegerin(OPG)/ receptor activator of NF-κB ligand(RANKL) in cultured rat osteoblasts. Methods Osteoblasts were isolated from skull of neonatal rats( 〈24 hours) by enzyme digestion, and fluorine of different concentrations [0 (control), 1×10^-3、1×10^-4、1×10^-5、1×10^-6 and 1×10^-7mol/L] were added into the culture medium of second generation of osteoblasts. The expressions of OPG and RANKL mRNA were determined using real-time PCR 24 and 48 hours after culturing. The expressions of OPG and RANKL protein were measured by Western blotting. Results (1) After exposed to fluorine for 24 hours, the differences of RANKL and OPG mRNA expression had statistical significance between groups(F = 30.95, 22.62, all P 〈 0.01), the expression of RANKL mRNA(5.99 ± 0.39) in the 1 × 10-5 mol/L group and the expressions ofOPG mRNA(3.52 ± 0.09, 4.81 ± 0.15, 3.68 ± 0.04) in the 1 × 10-4, 1 × 10-5 and 1 × 104 mol/L groups were higher than those of the control group(3.20 ± 0.19, 3.09 ± 0.58, all P 〈 0.05), but in the 1×10-3 mol/L group, RANKL mRNA(2.29 ± 0.18) was lower than that of the control group(P 〈 0.05). After exposed to fluorine for 48 hours, the differences of RANKL and OPG mRNA expression had statistical significance between groups(F = 26.62, 5.72, all P 〈 0.01), the expressions of RANKL and OPG mRNA(6.67 ± 0.49 and 5.05 ± 0.51) in the 1 × 10-5 mol/L group were higher than those of the control group(4.29 ± 0.07 and 4.34 ± 0.12, all P 〈 0.05), and in the 1 × 10-3 mol/L group the expression of OPG mRNA(3.63 ± 0.49) was lower than that of the control group(P 〈 0.05). (2) The expression of RANKL protein was not statistically significant between 24 hours and 48 hours groups(F = 0.07, 0.49, all P 〉 0.05) ; the differences of OPG protein expression had statistical significance between groups(F = 3.26; P 〈 0.05), the expression of OPG prote