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.
Objective 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.
出处
《中国地方病学杂志》
CAS
CSCD
北大核心
2010年第3期267-272,共6页
Chinese Jouranl of Endemiology
基金
国家自然科学基金项目（30560131）
关键词
氟中毒
基因表达
骨转化
Fluorosis
Gene expression
Bone turn over
