目的探讨细胞内钙离子水平([Ca^2+]i)和活性氧(ROS)在氟化钠(NaF)致人神经母细胞瘤SH—SY5Y细胞损伤中的关系。方法用40mg/LNaF对SH—SY5Y细胞进行染毒,检测在染毒不同时间(0、3、6、12、18、24h)的[Ca^2+],和ROS水平的变化,选择最佳染毒时间;观察最佳染毒时间(12h)40mg/LNaF与38.23mg/LBAPTA—AM或380.40mg/L乙二醇双(2-氨基乙醚)四乙酸(EGTA)或16.32mg/LN-乙酰半胱氨酸(NAC)共同和单独作用下,[Ca^2+]i、细胞内ROS和培养液乳酸脱氢酶(LDH)水平变化。结果染毒3、6、12、18、24h,[Ca^2+]i水平(5620.0±226.3、4775.5±85.6、3312.3±87.5、3047.0±75.0、2717.0±66.5),与0h(2115.0±24.0)比较,均明显升高(P〈0.05);ROS水平(4449.53±324.61、7463.07±117.43、20227.33±178.04、8817.56±200.13、7975.61±92.90),与0h(4098.01±21.22)比较,除3h外也明显升高(P〈0.05);[Ca^2+]i、ROS达到峰值的时间分别为3、12h。[Ca^2+]i、LDH水平,NaF组[3279.5±94.0,(1057.50±64.35)U/L]、NaF+NAC组[3583.0±350.7,(561.02±85.50)U/L、NaF+EGTA组[3701.5±157.7,(1074.50±86.97)U/L]、NaF+BAPTA—AM组[2766.5±38.9,(521.43±40.80)U/L],与对照组[2022.5±118.1,(186.97±8.73)U/L]比较,均有明显升高(P〈0.05);ROS水平,NaF组(19003.04±332.34)、NaF+EGTA组(19170.12±95.46)明显高于对照组(4060.98±145.66,P〈0.05)。在对ROS和LDH的影响中,NaF与NAC之间存在拮抗作用(F值分别为976.11、43.54,P〈0.05)。在对[Ca^2+]i、ROS和LDH水平的影响中,NaF与BAPTA—AM之间存在拮抗作用(F值分别为15.65、1515.53、115.00,P〈0.05)。结论NaF诱导SH—SY5Y细胞损伤的机制可能是通过胞内钙库的释放而提高[Ca^2+]i水平,升高的[Ca^2+]i促使ROS?
Objective To explore the relationship between intracellular calcium levels ( [ Ca^2+ ]i ) and reactive oxygen species (ROS) in sodium fluoride (NaF)-induced injury in human neuroblastoma SH-SY5Y cells. Methods The levels of [ Ca^2+ ]i and ROS were measured in different exposed times(0,3,6,12,18,24 h) respectively after SH-SY5Y cells were exposed to 40 mg/L NaF in vitro, and the optimal expose time was selected. Furthermore, the changes of [ Ca^2+ ]i, ROS and LDH levels in 40 mg/L NaF-treated groups ineubated with 38.23 mg/L BAPTA-AM or 380.40 mg/L ethylene glycol-bis-(beta-aminoethyl ether)-N,N,N' ,N'-tetraacetic acid (EGTA) or 16.32 mg/L N- aeetyl-L-cysteine(NAC) also observed at the optimal expose time(12 h), respectively. Results At 3,6,12,18 and 24 h, [ Ca^2+ ]i level(5620.0±226.3,4775.5 ± 85.6,3312.3 ± 87.5, 3047.0 ± 75.0,2717.0 ± 66.5) was significantly increased, and so was the ROS level (4449.53 ± 324.61,7463.07 ± 117.43,20 227.33 ± 178.04,8817.56 ± 200.13, 7975.61± 92.90) except at 3 h, compared with 0 h(2115.0 ±24.0,4098.01 ± 21.22, all P 〈 0.05). The levels of [ Ca^2+ ]i and ROS reached the peak at 3 h and 12 h, respectively. [ Ca^2+ ]i and LDH levels in NaF-treated group [3279.5± 94.0, (1057.50±64.35)U/L], NaF + NAC treated group[3583.0 ± 350.7, (561.02 ± 85.50)U/L], NaF + EGTA treated groups[3701.5 ± 157.7, (1074.50 ±86.97)U/L], and BAPTA-AM treated group[2766.5 ±38.9, (521.43 ± 40.80)U/L] had increased, compared with the control[2022.5± 118.1, (186.97 ± 8.73)U/L], the difference being statistically significant (P 〈 0.05). ROS levels in NaF-treated group ( 19 003.04 ±332.34 ), and NaF + EGTA treated group ( 19 170.12 ± 95.46) was higher than that in the controls (4060.98 ±145.66), the difference being statistically significant (P 〈 0.05). NaF and NAC had antagonistic effect on ROS and LDH levels (F = 976.11,43.54 ,P 〈 0.05 ). And NaF and BAPTA-AM had antagonis