过量氟诱导成釉细胞钙超载及细胞凋亡的实验研究

目的 研究过量氟对体外培养大鼠成釉细胞内钙超载及细胞凋亡的影响。方法 取大鼠成釉细胞系HAT-7细胞，分别加入不同浓度（0、0.4、0.8、1.6、3.2、6.4 mmol·L^-1）的氟化钠培养液，培养48 h后，采用Cell Counting Kit 8（CCK-8）试剂盒检测各组细胞的活性，流式细胞术分析氟对细胞凋亡的影响，激光扫描共聚焦显微镜、Western blot试验和实时荧光定量聚合酶链反应技术检测过量氟诱导大鼠成釉细胞内Ca²⁺浓度和钙网蛋白表达的变化。结果 氟化钠浓度高于1.6 mmol·L^-1时，可抑制成釉细胞的活性，成釉细胞内Ca²⁺浓度升高，钙网蛋白表达上调，细胞早期凋亡数量增加，并且随着浓度的增加，细胞凋亡的数量也随之增加。结论 过量氟可引起成釉细胞内钙超载，诱导成釉细胞凋亡。

Excessive fluoride inducing calcium overload and apoptosis of ameloblasts

Objective To study the effect of excessive fluoride on calcium overload and apoptosis in cultured rat ameloblasts in vitro. Methods Logarithmic-phase ameloblasts (HAT-7) were treated with 0, 0.4, 0.8, 1.6, 3.2, and 6.4 mmol·L^-1 sodium fluoride (NaF) solution. Cell activities were detected by using a Cell Counting Kit 8 (CCK-8) assay after 48 h of treatment. The effect of fluoride on cell apoptosis was analyzed by using flow cytometry. Excessive fluoride-induced calcium concentration and calreticulin expression changes in ameloblasts were detected by using laser scanning confocal microscopy, Western blot analysis, and real-time quantitative polymerase chain reaction. Results NaF inhibited ameloblast activity at 1.6, 3.2, and 6.4 mmol·L⁻¹ (dose-dependent) after 48 h of induction. The Ca²⁺ fluorescence intensity of HAT-7 cells incubated with 1.6 and 3.2 mmol·L⁻¹ NaF was higher than that in the control group. The fluoride-induced early-stage apoptosis of ameloblasts after 48 h of induction and the early-stage apoptosis rate was positively correlated with fluoride concentration. Calreticulin mRNA expression in HAT-7 cells was higher than that in the control group after 48 h of incubation with 0.8, 1.2, and 1.6 mmol·L⁻¹ NaF. Conclusion Excessive fluoride-induced calcium overload in ameloblasts and further caused endoplasmic reticulum stress-mediated apoptosis.