氟对小鼠成骨细胞增殖与分化及骨保护蛋白和核因子κβ受体活化因子配体mRNA 表达的影响

目的 观察氟对体外培养的小鼠成骨细胞增殖、分化、骨保护蛋白(OPG)mRNA及核因子κβ受体活化因子配体(RANKL)mRNA表达的影响.方法 取出生1～2 d昆明小鼠颅盖骨,分离成骨细胞后进行传代培养.按培养液中加入不同浓度氟化钠(NaF)将成骨细胞分为0(对照)、10-8、10-7、10-6、10-5、10-4、10-3 mol/L组,分别在培养72 h或120 h后检测氟对成骨细胞增殖、分化(碱性磷酸酶,ALP)的影响;提取成骨细胞总mRNA,采用RT-PCR方法分析成骨细胞OPG mRNA、RANKL mRNA表达,并进行半定量分析.组间比较采用单因素方差分析,两两比较采用LSD-t检验.结果 培养72 h后,成骨细胞增殖组间比较差异有统计学意义(F=13.806,P<0.05);与对照组(0.434±0.010)比较,10-7～10-4mol/L组成骨细胞增殖明显增加(0.448±0.010、0.453±0.013、0.454±0.016、0.449±0.018,P均<0.05),10-3 mol/L组成骨细胞增殖降低(0.401±0.009,P<0.05).成骨细胞ALP活性组问比较差异有统计学意义(F=9.021,P<0.05);其中10-7～10-5 mol/L组[(2.447±0.756)×106、(2.603±0.183)×106、(2.687±0.886)×106 U/L]ALP活性明显高于对照组[(1.677±0.682)×106U/L,P<0.05或<0.01];10-4mol/L组[(1.479±0.366)×106 U/L]ALP活性明显低于对照组(P<0.05).OPG mRNA表达组间比较差异有统计学意义(F=11.299,P<0.05);与对照组(11000±0.000)比较,10-7～10-4 mol/L组表达增强(1.058±0.027、1.053 ±0.026、1.088±0.055、1.069±0.008,P<0.05或<0.01),10-3mol/L组表达降低(0.941±0.029,P<0.05).成骨细胞RANKL mRNA表达组间比较差异无统计学意义(F=1.311,P>0.05).RANKL mRNA/OPG mRNA比值,在104～10-5mol/L组逐渐降低,10-4、10-3mol/L组升高,但组间比较差异无统计学意义(F=1.376,P>0.05).结论 氟对小鼠成骨细胞增殖、分化呈双向调节作用,表现为低剂量刺激而高剂量抑制.低剂量染氟可能通过增加成骨细胞OPG的表达来抑制破骨细胞的分化和成熟,抑制骨吸收;而高剂量可能通过降低OPG的表达来促进破骨细胞的分化、成熟,促进骨吸收.

Abstract：

Objective To investigate the effect of sodium fluoride(NaF) on proliferation, differentiation and the mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor κβ ligand (RAN KL) of mouse osteoblasts. Methods Osteoblasts were isolated from calvarias of Kunming mice born in 1 - 2 d and cultured. Various concentrations of NaF(0, 10-8, 10-7, 10-6, 10-5, 10-4, 10-3mol/L) were added to the culture medium, the proliferation and activity of alkaline phosphatase(ALP) was determined after 72 h or 120 h. The expression of OPG mRNA and RANKL mRNA was analyzed by semi-quantification RT-PCR. Difference among groups was analyzed by One-Way AN0VA. Difference between two groups was analyzed by LSD-t test. Results There was significant difference in cell proliferation among groups after 72 h(F = 13.806, P < 0.05). Compared with control group(0.434 ± 0.010) , the proliferation was significantly induced in 10-7 - 10-4 mol/L groups treated osteoblasts (0.448 ± 0.010, 0.453 ± 0.013, 0.454 ± 0.016, 0.449 ± 0.018, all P< 0.05), and was significantly suppressed in 10-3 mol/L group(0.401 ± 0.009, P < 0.05). There was statistic difference in the activity of ALP among groups(F = 9.021, P < 0.05). Compared with control group (1.677 ± 0.682), the activity of ALP significantly increased in 10-7 - 10-5 mol/L groups[ (2.447 ± 0.756) × 106, (2603 ± 0.183) × 106, (2.687 ± 0.886) × 106 U/L, P < 0.05 or P < 0.01 ] and significantly decreased in 10-4 mol/L group[ (1.479 ± 0.366) × 106 U/L, P < 0.05 ]. There was significant difference in the expression of OPG mRNA among groups(F = 11.299, P< 0.05). Compared with control group (1.000 ± 0.000), the expression of OPG mRNA was significantly increased in 10-7 - 10-4 mol/L groups( 1.058 ± 0.027, 1.053 ± 0.026, 1.088 ± 0.055, 1.069 ± 0.008, P < 0.05 or P < 0.01) , while significantly decreased in 10-3 mol/L group (0.941 ± 0.029, P< 0.05). There was no difference in RANKL mRNA expression among groups (F= 1.311, P> 0.05). The ratio of RANKL/OPG decreased with increasing doses of fluoride and increased in 10-4, 10-3 mol/L groups, but there was no difference between groups(F = 1.376, P> 0.05). Conclusions A biphasic pattern of proliferation and differentiation has been induced in mouse osteoblasts, which manifests stimulation effect in low doses and suppression in higher doses. Low doses of sodium fluoride suppress differentiation and maturation of osteoblasts by increasing expression of OPG mRNA, while high doses of sodium fluoride enhance differentiation and maturation of osteoblasts by decreasing expression of OPG mRNA.