氟对大鼠切牙细胞Fas信号通路的作用研究

Effects of fluoride on Fas signal pathway in rat incisor cells

目的研究氟对大鼠切牙细胞Fas表达及胱天蛋白酶（caspase）3和easpase-8活性和细胞凋产的影响，以期进一步探讨氟对牙齿的作用以及氟斑牙的发生机制。方法将40只SD大鼠按随机数字表法随机分为4组，每组10只。低剂量染氟组（低氟组）、中剂量染氟组（中氟组）和高剂量染氟组（高氟组）分别饮用以蒸馏水配制的含10、50和100mg／LNaF的高氟水；对照组饮用不添加NaF的蒸馏水；在60、90d时各组分别处死5只动物。用免疫组化法检测Fas的表达，采用酶标仪检测easpase-3和caspase-8的活性，用流式细胞术检测大鼠切牙细胞凋亡。结果在染氟60d时，对照组、低氟组、中氟组及高氟组的Fas表达结果分别为：0．1819±0．0025、0．2120±0．0084、0．2283±0．0183及0．2818±0．0233；在染氟90d时，对照组、低氟组、中氟组及高氟组的Fas表达结果分别为：0．2077±0．0289、0．2216±0．0105、0．2377±0．0059及0．2775±0．0088。大鼠切牙细胞Fas表达和caspase-3活力随染氟剂量增高而增强，存在显著的剂量-效应关系，实验60及90d时Fas的相关系数分别为0．9728（P〈0．01）和0．9889（P〈0．01）；easpase-3的相关系数分别为0．9533（P〈0．01）和0．9849（P〈0．01）。在染氟90d时，大鼠切牙细胞凋亡率和caspase-8活力随染氟剂量增高而增强，凋亡率和easpase-8的相关系数分别为0．9733（P〈0．01）和0．9928（P〈0．01）。在染氟60和90d时，大鼠切牙细胞Fas表达与easpase-3活力之间存在明显相关关系，相关系数分别为0．9619（P〈0．01）和0．9912（P〈0．01）；在染氟90d时，大鼠切牙细胞Fas表达与凋亡率和caspase-8活力之间也有明显的相关关系，Fas与凋广率和easpase-8的相关系数分别为0．9841（P〈0．01）和0．9767（P〈0．01）。结论在10、50和100mg／LNaF剂量条件下染氟60和90d，大鼠切牙细胞Fas表达增强并介导caspase激活和细胞凋产，详尽机制仍需深入探讨。

Objective To investigate the effects of fluoride on Fas expression, caspase-3 and easpase-8 activity and apoptosis in rat incisor cells. Methods Forty male SD rats were divided into 4 groups randomly and provided with distilled water containing NaF at the doses of 0, 10, 50 and 100 mg/L respectively. Each group had lO animals. Five animals were sacrificed at 60 and 90 days respectively. Fas expression was measured with immunohistochemistry, and colorimetric assay was used to examine caspase-3 and caspase-8 activity with enzyme-labelled meter. The apoptosis was detected by flow eytometry in mandibular incisor cells. Results NaF at the doses of 10,50 and 100 mg/L for 60 d and 90 d caused Fas overexpression, promoted activity of caspase-3 and caspase-8, increased apoptosis rate in mandibular incisor cells. At 60 days, the value of Fas expression was 0. 1819 ± 0. 0025 for control, 0. 2120 ± 0. 0084 for 10 mg/L NaF group, 0. 2283±0. 0183 forS0 mg/L NaF group, 0. 2818±0. 0233 for 100 mg/L NaF group. At 90 days,the value of Fas expression was O. 20W±0. 0289 for control, O. 2216±0. 0105 for 10 mg/L NaF group, 0. 2377 ± O. 0059 for 50 mg/L NaF group, 0. 2775 ± 0. 0088 for 100 mg/L NaF group. Statistics analysis yielded close relationship between the dose of NaF in water and the Fas expression, and alsobetween the dose of NaF in water and caspase-3 activities, and the relative coefficient was O. 9728 （ 60 d, P 〈 O. O1 ） and O. 9889 （90 d, P 〈 O. 01 ） for Fas expression, O. 9533 （ 60 d, P 〈 0. 01 ） and 0. 9849 （ 90 d, P 〈 O. O1 ）for caspase-3 activity respectively. Apoptosis rate and caspase-8 activity also had close relationship with the NaF doses, and the relative coefficient was 0. 9733 （90 d, P 〈 0. 01 ）for apoptosis, O. 9928 （90 d, P 〈0. 01 ）for caspase-8. At the doses of 10, 50 and 100 mg/L NaF for 60 d and 90 d, obvious relationship was found between Fas expression and caspase-3 activity, and the relative coefficient was 0. 9619 （60 d, P 〈 O. 01） and O. 9912（90 d, P 〈0. 01 ）. Obvious relationship between Fas expression and apoptosis, between Fas expression and caspase-8 activity was found in groups for 90 d, and the relative coefficient was 0. 9841 （P 〈 O. O1 ） for apoptosis, O. 9767 （ P 〈 0.01 ） for caspase-8. Conclusions Fluoride could induce Fas overexpression and mediate caspase activation and apoptosis at the doses of 10,50 and 100 mg/L for 60 d and 90 d in rat mandibular incisor cells.