成纤维细胞生长因子23在氟中毒小鼠骨组织中的表达

Role of fibroblast growth factor-23 in bone injury induced by fluoride in mice

目的 观察氟对小鼠骨组织中成纤维细胞生长因子23（FGF23）的影响，探讨FGF23在氟致小鼠骨相损伤中的作用。方法 64只Balb／c小鼠，雌雄各半，按体质量采用随机数字表法分成4组，每组16只。对照组、低氟组、中氟组、高氟组分别给予含0、25、50、100 mg／L氟离子的蒸馏水，3个月后处死，观察小鼠氟斑牙患病情况。用氟离子选择电极法测脊椎骨骨氟含量，微量酶标仪法测血清中钙、磷含量，酶联免疫吸附法测血清中FGF23、甲状旁腺素（PTH）、1，25二羟基维生素D3［1，25（OH）2D3］含量；免疫组织化学法、蛋白免疫印迹法（Western blot）分别测骨组织中FGF23蛋白表达水平。结果 低、中、高氟组小鼠氟斑牙检出率分别为75%（4／16）、100%（16／16）、100%（16／16），与对照组［0（0／16）］比较，差异均有统计学意义（P均 〈 0.05）。各染氟组骨氟水平［低、中、高氟组：（1 730.86 ± 165.90）、（2 400.58 ± 286.65）、（3 980.88 ± 511.65）mg／kg］均高于对照组［（854.30 ± 89.05）mg／kg，P均 〈 0.01］。血清中钙含量组间比较差异无统计学意义（F = 0.05, P 〉 0.05），中、高氟组血清中磷含量［（2.46 ± 0.32）、（2.48 ± 0.73）mmol／L］与对照组、低氟组［（2.89 ± 0.45）、（3.25 ± 0.69）mmol／L］比较明显降低（P均 〈 0.05）。血清中PTH、1，25（OH）2D3含量均呈现先升高后降低的趋势；中、高氟组FGF23的表达［（660.84 ± 64.18）、（638.74 ± 121.23）ng／L］与对照组［（613.53 ± 98.18）ng／L］比较有升高趋势。免疫组化结果显示，在皮质骨中FGF23蛋白表达水平随染氟剂量增加逐渐升高。Western blot结果显示，小鼠骨组织中FGF23蛋白含量在各染氟组中均明显增加，且低氟组（1.58 ± 0.46）、中氟组（1.40 ± 0.41）与对照组（1.00 ± 0.41）比较，差异均有统计学意义（P均 〈 0.05）。结论 氟中毒小鼠血清中磷、FGF23、PTH、1，25（OH）2D3含量及骨组织中FGF23蛋白表达水平均有变化，提示FGF23可能通过与PTH、1，25（OH）2D3相互作用，影响体内钙磷代谢水平而参与氟骨症的形成。

Objective To observe the effect of fluoride on fibroblast growth factor 23 （FGF23） in bone tissue of mice, and to explore the role of FGF23 in fluoride-induced bone injury. Methods Sixty-four Balb/c mice, half male and female, were divided into 4 groups based on body weight via the random number table method and 16 mice were in each group. The mice in control group, low fluoride group, middle fluoride group and high fluoride group were treated with 0, 25, 50, and 100 mg/L F- distilled water, respectively. After three months, the mice were put to death and the prevalence of dental fluorosis was calculated. The fluoride contents in spine were detected via the fluoride-ion selective electrode method, serum content of calcium and phosphorus were detected by micro enzyme labeled method. The levels of FGF23, parathyroid hormone （PTH） and 1,25 dihydroxy vitamin D3 [1,25 （OH）2D3] in serum were measured by enzyme-linked immunosorbent assay. The FGF23 protein expression levels in bone tissue were determined by immunohistochemistry and Western blotting. Results The rates of dental fluorosis in low, medium and high fluoride groups were 75% （4/16）, 100% （16/16） and 100% （16/16）, respectively. Compared with control group [0 （0/16）] the differences were statistically significant （P 〈 0.05）. The levels of fluoride in the fluoride group [low, medium, high fluoride groups： （1 730.86 ± 165.90）, （2 400.58 ± 286.65）, （3 980.88 ± 511.65） mg/kg] were higher than that of control group [（854.30 ± 89.05） mg/kg, P 〈 0.05]. There was no difference in serum calcium content among groups （F = 0.05, P 〉 0.05）. The contents of phosphorus in the serum of the medium and the high fluoride groups [（2.46 ± 0.32）, （2.48 ± 0.73） mmol/L] were lower than those in the control and the low fluoride groups [（2.89 ± 0.45）, （3.25 ± 0.69） mmol/L, P 〈 0.05]. The serum PTH and 1,25 （OH）2D3 content increased first and then decreased. The expression of FGF23 in middle and high fluoride groups [（660.84 ± 64.18）, （638.74 ± 121.23） ng/L] was up-regulated compared with that of control group [（613.53 ± 98.18） ng/L]. The expression of FGF23 protein in cortical bone increased gradually with the dose of fluoride. Western blotting results showed that the content of FGF23 protein in the bone tissue of mice was significantly increased in the low fluoride group （1.58 ± 0.46） and the middle fluoride group （1.40 ± 0.41） compared with that of control group （1.00 ± 0.41）, the differences were statistically significant （P 〈 0.05）. Conclusions The phosphorus, FGF23, PTH, and 1,25 （OH）2D3 levels in the serum and FGF23 protein levels in the bone tissue of fluorosis mice have changed. It may be suggested that FGF23 interacts with PTH and 1,25 （OH）2D3 to influence the level of calcium and phosphorus metabolism in the body and participate in the formation of skeletal fluorosis.