氟化钠诱导人神经母细胞瘤SH-SY5Y细胞凋亡及其机制研究  

Apoptosis of Human Neuroblastoma SH-SY5Y Cells Induced by Sodium Fluoride

作　　者：徐八一[1] 王普生 黄建云[1] 徐志霞[1] 夏涛[2] 王爱国[2] 

机构地区：[1]汕头市疾病预防控制中心疾病控制科,广东汕头515041 [2]华中科技大学同济医学院公共卫生学院劳动卫生与环境卫生学系教育部环境与健康重点实验室

出　　处：《环境与健康杂志》2011年第11期944-946,共3页Journal of Environment and Health

基　　金：国家自然科学基金(81072266);广东省医学科研基金(B2009234)

摘　　要：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 study the neurons damage and apoptosis induced by sodium fluoride （NaF） in human neuroblastoma SH-SY5Y cells and to explore the the potential mechanism of neurotoxicity of NaF. Methods SH-SY5Y cells of logarithmic phase （1×10^7/ml） were first incubated with fluoride （0, 20, 40, 80 mg/L） and then with ethyleneglycol-bis-（beta- aminoethyl ether） -N,N,N＇ ,N＇ -tetraacetic acid （EGTA, 380.40 mg/L）, 1,2 -bis （O -aminophenoxy）ethane -N,N,N＇ ,N＇ - tetraacetic acid tetra（acetoxymethyl） ester （BAPTA-AM, 38.23 mg/L） alone or combined with fluoride （40 mg/L） respectively for 24 h in vitro.The cell viability was measured by MTT （thiazolyl blue）. The percentage of apoptosis and intracellular calcium concentration [Ca2＋]i were measured by flow cytometry. Furthermore ,laser scanning eonfocal microscope （LSCM） was used to test the change of intracellular free calcium concentration in single SH-SY5Y cells. Results Compared with the control group,the cell survival rates in 40 and 80 mg/L NaF-treated groups were significantly lower （P〈0.05）. The percentage of apoptosis and [CaZ＋]i in 40 and 80 mg/L NaF-treated groups were higher （P〈0.05） than that in the control group,and increased as the dose of NaF increased. The study of laser scanning confocal microscope found that the increase of NaF concentration shortened the reaching peak time of [CaZ＋]i. Factorial analysis showed that intracellular calcium-chelating compound （BAPTA-AM） and NaF had interaction on apoptosis and the elevation of [Ca2＋]i （F=10.69,366.14,P〈0.05）, BAPTA-AM could antagonize the cell injury caused by NaF in SH-SY5Y cells.Extracellular calcium chelator EGTA and NaF had no interaction. Conclusion NaF exposure could cause the elevation of calcium from the site of intracellular calcium storage,contributing to the damage and apoptosis induced by NaF.

关 键 词：氟化钠 人神经母细胞瘤细胞株 细胞凋亡 钙离子 

分 类 号：R994.6[医药卫生—毒理学]