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目的研究氟化钠(NaF)对人神经母细胞瘤SH-SY5Y细胞的损伤及致凋亡作用,并初步探讨NaF致神经毒性的作用机制。方法取对数生长期的SH-SY5Y细胞(1×107/ml),先加入0(对照)、20、40、80 mg/L的NaF溶液对SH-SY5Y细胞染毒24 h;再选择40 mg/L的NaF溶液与380.40 mg/L的乙二醇双(2-氨基乙醚)四乙酸(EGTA)溶液或38.23 mg/L的胞内钙离子螯合剂(BAPTA-AM)溶液(提前30 min加入)联合染毒24 h。采用四甲基偶氮唑盐(MTT)法检测细胞活性,采用流式细胞仪检测细胞凋亡率和胞内钙离子([Ca2+]i)水平,并用激光共聚焦(laser scanningconfocal microscopy,LSCM)技术,从单个细胞水平检测NaF对胞内游离Ca2+瞬间动态变化的影响。结果与对照组比较,40、80 mg/L NaF染毒组SH-SY5Y细胞的存活率较低,凋亡率较高,差异均有统计学意义(P<0.05);各NaF染毒组SH-SY5Y细胞的[Ca2+]i水平均高于对照组,差异有统计学意义(P<0.05)。且随着NaF染毒浓度的升高,SH-SY5Y细胞的存活率呈下降趋势,凋亡率和[Ca2+]i水平均呈上升趋势。LSCM连续扫描显示,40和80 mg/L NaF染毒组SH-SY5Y细胞[Ca2+]i的FI值达到峰值的时间分别为6 000～7 000 s和1 000～2 000 s,20 mg/L NaF染毒组未见明显的峰值;且NaF的浓度越高,[Ca2+]i达到峰值的时间越短。析因分析表明,胞内钙离子螯合剂(BAPTA-AM)与NaF对SH-SY5Y细胞凋亡和[Ca2+]i升高存在交互作用(F值分别为10.69、366.14,均P<0.05),BAPTA-AM可拮抗NaF对SH-SY5Y细胞的损伤作用;胞外钙离子螯合剂乙二醇双四乙酸(EGTA)与NaF对SH-SY5Y细胞凋亡和[Ca2+]i升高无交互作用(F值分别为0.02、0.03,均P>0.05)。结论 NaF对SH-SY5Y细胞的升钙作用可能参与了NaF的致细胞损伤和诱导凋亡作用,[Ca2+]i的升高可能与胞内钙库的释放有关。
Objective To investigate the effect of sodium fluoride (NaF) on the injury and apoptosis of human neuroblastoma SH-SY5Y cells and to explore the mechanism of NaF-induced neurotoxicity. Methods SH-SY5Y cells in logarithmic growth phase (1 × 107 / ml) were treated with 0 (control), 20,40,80 mg / L NaF solution for 24 h. Then 40 mg / L NaF solution and 380.40 mg / L EGTA solution or 38.23 mg / L BAPTA-AM solution (30 min in advance) Join) joint exposure 24 h. The cell viability was detected by MTT assay. The apoptosis rate and intracellular calcium ([Ca2 +] i) level were detected by flow cytometry. Laser scanning confocal microscopy (LSCM) Technology to detect the effect of NaF on transient intracellular free Ca2 + changes from a single cell level. Results Compared with the control group, the survival rates of SH-SY5Y cells in 40 and 80 mg / L NaF groups were lower and the apoptosis rates were higher (P <0.05) -SY5Y cells [Ca2 +] i levels were higher than the control group, the difference was statistically significant (P <0.05). With the increase of NaF concentration, the survival rate of SH-SY5Y cells decreased and the apoptosis rate and [Ca2 +] i level increased. LSCM serial scans showed that the FI values of [Ca2 +] i in SH-SY5Y cells in 40 and 80 mg / L NaF exposure groups were 6 000 ~ 7 000 s and 1 000 ~ 2 000 s, 20 mg / L, respectively No significant peak was observed in NaF-treated group; the higher the concentration of NaF, the shorter the peak of [Ca2 +] i. Factorial analysis showed that there was interaction between intracellular calcium chelator (BAPTA-AM) and NaF on apoptosis and [Ca2 +] i in SH-SY5Y cells (F = 10.69 and 366.14, P <0.05, respectively) BAPTA-AM antagonized NaF-induced injury in SH-SY5Y cells. EGTA and NaF had no effect on the apoptosis of SH-SY5Y cells and the increase of [Ca2 +] i F values were 0.02,0.03, all P> 0.05). Conclusion NaF may play a role in inducing cell apoptosis and inducing apoptosis in SH-SY5Y cells. The increase of [Ca2 +] i may be related to the release of intracellular calcium stores.