摘要目的 观察氟中毒大鼠成骨细胞Wnt3a、β-链蛋白(catenin)mRNA和蛋白表达,探讨氟骨症发生与Wnt通路的关系.方法 健康SD大鼠36只,体质量100～120 g,按体质量将大鼠随机分为3组,每组12只.对照组大鼠饮用自来水(含氟量＜1 mg/L),低氟组、高氟组大鼠分别饮用含5、50 mg/L氟化钠的自来水.大鼠饲养8个月,建立慢性氟中毒模型.饲养期间检查大鼠氟斑牙发生情况,股动脉放血处死大鼠前收集大鼠24 h尿样,处死后取股骨组织.采用氟离子选择电极法测定尿氟和骨氟含量；固相夹心酶联免疫吸附(ELISA)法测定血清骨碱性磷酸酶(BALP)和抗酒石酸酸性磷酸酶-5b(TRACP-5b)含量；光镜下观察骨组织的形态学变化,测量骨皮质厚度、骨小梁宽度及密度变化；原位杂交技术和免疫组化方法检测成骨细胞Wnt3a、β-catenin mRNA和蛋白表达.结果 大鼠氟斑牙检出率低氟组为66.7％(8/12),高氟组为91.7％(11/12),对照组为0.0％(0/12),组间比较差异有统计学意义(χ2=21.6,P＜0.05).尿氟和骨氟组间比较差异有统计学意义(F=36.57、467.02,P均＜0.05),其中低氟组[(2.06±0.64)mg/L、(632.33±123.21) mg/kg]和高氟组[(7.69±1.96)mg/L、(1088.75±156.16)mg/kg]高于对照组[(1.26±0.17)mg/L、(305.58±91.26)mg/kg,P均＜0.05],高氟组高于低氟组(P均＜ 0.05).血清BALP和TRACP-5b组间比较差异有统计学意义(F=89.57、7.68,P均＜0.05).其中低氟组[(31.47±5.30)U/L]和高氟组[(54.61±2.27)U/L].血清BALP明显高于对照组[(16.24±1.57)U/L,P均＜0.05],高氟组高于低氟组(P＜0.05)；血清TRACP-5b,低氟组[(3.45±1.85)U/L)明显高于对照组[(1.26±0.23)U/L]和高氟组[(2.74±1.8)]U/L,P均＜0.05].光镜下,高氟组和低氟组大鼠股骨骨皮质较对照组增厚,骨小梁增宽、排列紧密.原位杂交和免疫组化显示,近骨小梁表面的成骨细胞细胞质和细胞核呈棕黄色阳性着色.图像分析显示,Wnt3a、β-catenin mRNA和蛋白表达组间比较差异有统计学意义(F值分别为12.47、5.96,10.07、53.82,P均＜0.05).其中低氟组(132.87±5.72、132.57±9.56,137.50±4.32、140.85±3.54)和高氟组(135.60±6.64、137.87±9.16,142.65±11.84、152.52±4.64)均高于对照组(119.86±5.04、120.58±7.84,124.01±2.63、126.75±4.65,P均＜0.05)；除β-catenin蛋白表达高氟组明显高于低氟组(P＜0.05)外,其他两组间比较差异无统计学意义(P均＞0.05).相关分析显示,Wnt3amRNA与β-catenin mRNA表达、Wnt3a蛋白与β-catenin蛋白表达,二者之间都具有相关性(r值分别为0.731、0.658,P均＜0.05).结论 过量氟引起的大鼠骨组织的病变可能与Wnt3a、β-catenin mRNA和蛋白在成骨细胞的表达增高有关.慢性氟中毒时,氟通过刺激Wnt经典信号通路中Wnt3a、β-catenin的过表达,使成骨作用增强,从而引起骨骼的病变而发生氟骨症.

abstractsObjective To explore the effect of excessive fluoride on expression of mRNA and protein of Wnt3a and β-catenin in rats' osteoblasts and its correlation with pathogenic mechanism of fluorosis.Methods Thirty-six healthy SD rats,weighting 100-120 g and according to body mass,were randomly divided into three groups(twelve in each group).The rats of control were fed wich tap water(fluoride ＜ 1 mg/L) and the experimental rats were exposed to NaF(low-fluoride group:5 mg/L,high-fluoride group:50 mg/L) added to the drinking water to establish the chronic fluorosis model.After fed for eight morth,all rats were killed and metaphysic of femoral was collected.Rat dental fluorosis was observed and bone fluorine was detected by ashing-fluorin ion selective electrode method.The content of bone alkaline phosphatase (BALP) and tartrate-resistant acid phosphatase 5b(TRACP 5b) in rats' serum was detected by enzyme-linked immunosorbent assay (ELISA).The morphologic changes of the bone were observed by microscopy.The expression of mRNA and protein of Wnt3a and β-catenin in osteoblasts of rats was analyzed with gray scale by hybridization in situ and immunohistochemistry methods,respectively.Results Detection rate of dental fluorosis,fluoride contents of urine and bone were significantly increased [control group:0.0％,(1.26 + 0.17)mg/L,(305.58 ± 91.26)mg/kg; low-fluoride group:66.7％,(2.06 ± 0.64)mg/L,(632.33 ±123.21)mg/kg; high-fluoride group:91.7％,(7.69 ± 1.96)mg/L,(1088.75 ± 156.16) mg/kg] in the rats treated with fluoride,the difference between groups was statistically significant(χ2 =21.6; F =36.57,467.02; all P ＜0.05).The contents of BALP and TRACP-5b in rats' serum were significantly different between groups(F =89.57,7.68; all P ＜ 0.05).Compared with control group[(16.24 + 1.57)U/L],the contents of BALP in rats' serum of the low-fluoride and high-fluoride groups[(31.47 ± 5.30) and (54.61 ± 2.27)U/L] were increased gradually(all P ＜0.05).Compared with the low-fluoride group,the value in the high-fluoride group decreased significantly (P ＜ 0.05).The contents of TRACP-5b in rats' serum of low-fluoride group[(3.45 ± 1.85)U/L] were elevated significantly(all P ＜ 0.05) compared with the control group[(1.26 ± 0.23)U/L] and the high-fluoride group[(2.74 ± 1.85)U/L].The bone cortices were thickened and the bone trabecula was broadened,arranged closely together in chronic fluorosis rats with significant difference compared with the control group.In the low-fluoride and high-fluoride groups,the expression levels of Wnt3a and β-catenin mRNA (low-fluoride group:132.87 ± 5.72 and 132.57 ± 9.56; highfluoride group:135.60 ± 6.64 and 137.87 ± 9.16) were markedly elevated with significant difference,respectively (F =12.47,5.96; all P ＜ 0.05) compared with those in control groups(119.86 ± 5.04 and 120.58 ± 7.84) by hybridization in situ(P ＜ 0.05),but there was no statistical significance (P ＞ 0.05) of the level of Wnt3a and β-catenin mRNA between low-fluoride and high-fluoride groups.In the low-fluoride and high-fluoride groups,the protein expression of Wnt3a and β-catenin (low-fluoride group:137.50 ± 4.32 and 140.85 + 3.54; high-fluoride group:142.65 ± 11.84 and 152.52 ± 4.64) were markedly elevated with significant difference,respectively (F =10.07,53.82; all P ＜ 0.05) compared with those in control group (124.01 ± 2.63 and 126.75 ± 4.65) by immunohistochemistry(all P＜ 0.05),Wnt3a protein production in the low-fluoride group was increased without statistical significance compared with the high-fluoride group (P ＞ 0.05).But the protein production of β-catenin in the lowfluoride group was elevated with significant difference compared with the high-fluoride group(P ＜ 0.05).The mRNA and protein production of Wnt3a were positively correlated with the mRNA and protein production of β-catenin (r =0.731,0.658; all P ＜ 0.05).Conclusions Rat bone tissue lesions caused by excessive fluoride may be associated with an increased expression of Wnt3a and β-catenin mRNA and protein in osteoblasts.In chronic fluorosis,fluoride stimulates the overexpression of Wnt3a and β-catenin in the Wnt signal transduction pathway,enhances bone osteogenesis and causes skeletal fluorosis.