摘要目的 观察氟对体外培养人成骨肉瘤细胞内微小染色体维系蛋白(minichromosone maintenance,MCM)3和成骨相关基因表达的影响,探讨MCM3基因能否作为诊断及监测成骨细胞染氟过量的生物学标志.方法 采用McCoy5A培养液,体外培养人成骨肉瘤细胞(Saos-2).按染氟(NaF)剂量将骨肉瘤细胞分为O(对照)、0.625、1.250、2.500、5.000、10.000、20.000、40.000 mg/L组,染氟培养24 h后收集细胞,实时荧光定量(real-time.RT-PCR)PCR测定成骨细胞MCM3和骨涎蛋白(bone sialoprotein,BSP)、骨钙素(osteocalcin,OC)、骨桥蛋白(osteopontin,OP)3种成骨相关基因mRNA的表达,化学比色法测定碱性磷酸酶(alkaline phosphatase,ALP)活性,用双标准曲线法计算基因表达的相对比值.结果 成骨肉瘤细胞MCM3 mRNA表达在0.625、1.250、2.500、5.000、20.000、40.000 mg/L组(0.059±0.003、0.027±0.001、0.272±0.004、0.115±0.002、0.137±0.004、0.754±0.002)低于对照组(1.000±0.020,P均>0.05),但10.000 mg/L组(21.300±1.200)显著高于对照组(P<0.01),也高于其他染氟组(P均<0.01),组间比较差异有统计学意义(F=305.842,P<0.01).BSPmRNA表达在0.625、1.250、2.500、5.000、10.000 mg/L组(71.80±3.60、133.00±7.20、85.50±0.60、80.90±1.20、304.00±21.00)显著高于对照组(1.00±0.04,P均<0.01),尤其10.000 mg/L组明显高于其他染氟组(P均<0.01),组问比较差异有统计学意义(F=159.531,P<0.01).OC mRNA表达在0.625、1.250、2.500、5.000 mg/L组(110.00±12.00、143.00±2.10、90.60±4.10、23.70±1.20)高于对照组(1.00±0.01,P均<0.01),组间比较差异有统计学意义(F=158.734,P<0.01).OP mRNA表达,在0.625、1.250、2.500、5.000、10.000、20.000 mg/L组(167.00±11.20、111.00±12.10、72.50±3.50、134.00±14.00、42.30±2.40、45.20±3.30)高于对照组(1.00±0.04,P均<0.05或<0.01),组间比较差异有统计学意义(F=60.226,P<0.01).ALP的活性虽然在0.625～40.000mg/L组(6.0±0.4、5.8±0.1、5.7±0.4、7.7±1.1、19.2±2.4、8.5±3.0、18.1±4.2)与对照组(4.2±1.2)比较是增加的,但仅10.000、40.000 mg/L组明显高于对照组和其他染氟组(P均<0.01),组间比较差异有统计学意义(F=7.806,P<0.01).结论 MCM3表达不规律,不适合作为诊断及监测成骨细胞在过量氟作用下的生物标志;氟可能通过影响各成骨相关基因的表达而促进成骨分化.

abstractsObjective To study the effects of fluoride on minichromosone maintenance(MCM)3 mRNA and the bone formation-related gene:bone sialoprotein(BSP),osteocalcin(OC),osteopontin(OP)mRNA expression on human osteoblast cells.The expression of MCM3 was tested for diagnosis and surveillance value on osteoblast treated with excess fluoride.Methods Human osteoblast cell(Saos-2)was cultured in McCoy5A medium and treated with fluoride(sodium fluoride,NaF).There were eight groups including:0(control),0.625,1.250,2.500,5.000,10.000,20.000,40.000 mg/L groups.Expression of MCM3,BSP,OC,OP mRNA were detected by real-time PCR.Dual-standard curve method was used for analysis.ALPase was determined by measuring the absorbance using a micro titer plate reader. Results Expression of MCM3 mRNA was lower in the 0.625,1.250,2.500,5.000,20.000, 40.000 mg/L groups(0.059 ± 0.003,0.027 ± 0.001,0.272 ± 0.004,0.115 ± 0.002,0.137 ± 0.004,0.754 ±0.002, all P > 0.05) and was higher in10.000 mg/L group(21.300 ± 1.200, P < 0.01 ) than control group( 1.000 ±0.020), especially 10.000 mg/L group was higher than groups treated with fluoride(all P < 0.01 ), the differences among groups were significant(F = 305.842, P < 0.01 ). Expression of BSP mRNA was significantly higher in 0.625,1.250,2.500,5.000,10.000 mg/L groups(71.80 ± 3.60,133.00 ± 7.20,85.50 ± 0.60,80.90 ± 1.20,304.00 ± 21.00)than the control group( 1.00 ± 0.04), especially 10.000 mg/L group was higher than others groups treated with fluoride(all P < 0.01 ), the differences among groups were signifieant(F = 159.531, P < 0.01 ). Expressions of OC mRNA were higher in 0.625,1.250,2.500,5.000 mg/L groups(110.00 ± 12.00,143.00 ± 2.10,90.60 ± 4.10,23.70±1.20) than control group(1.00 ± 0.01, all P < 0.01), and the differences among groups were significant (F = 158.734, P < 0.01 ). Expression of OP mRNA were higher in 0.625,1.250,2.500,5.000,10.000,20.000 mg/L groups(167.00 ± 11.20, 111.00 ± 12.10,72.50 ± 3.50,134.00 ± 14.00,42.30 ± 2.40,45.20 ± 3.30) than the control group(1.00 ± 0.04, all P < 0.05 or < 0.01 ), the differences among groups were significant(F = 60.226, P < 0.01 ).Compared with control group(4.2 ± 1.2), the ALPase activity was increased in all groups treated with fluoride (6.0 ± 0.4,5.8 ± 0.1,5.7 ± 0.4,7.7 ± 1.1,19.2 ± 2.4,8.5 ± 3.0,18.1 ± 4.2), but only 10.000 mg/L and 40.000 mg/L groups were higher than control group and other groups treated with fluoride(all P < 0.01 ), the differences among groups were signifieant(F = 7.806, P < 0.01 ). Conclusions Irregular expression of MCM3 mRNA is not suitable as a diagnostic and monitoring biomarker of osteoblasts exposed to excessive fluoride. Fluoride may affect the osteoblast-related gene expression and to promote osteogenic differentiation.