铅暴露对神经胶质瘤细胞铜代谢的影响及其机制

Effect of lead exposure on copper metabolism in glioma cells and its mechanism

目的：研究铜转运蛋白1（CTR1）、铜转运 ATP 酶α多肽（ATP7A）在铅暴露诱导大鼠 C6胶质瘤细胞铜离子代谢紊乱及氧化应激反应中的作用。方法采用噻唑蓝法（MTT），通过 C6细胞暴露于醋酸铅0～100μmol·L -124和48 h 筛选适合剂量。C6细胞用醋酸铅10μmol·L -1分别处理24和48 h 后，分别采用黄嘌呤氧化酶法和硫代巴比妥酸比色法（TBA）检测铅对细胞 SOD 活性和 MDA 含量；原子吸收分光光度法观察铅对细胞内铜水平；荧光实时定量 PCR 和 Western blot 分别检测铅暴露后细胞 CTR1和ATP7A 的 mRNA 和蛋白表达水平的变化。结果醋酸铅浓度高于10μmol·L -1时，细胞活力受到显著抑制，故选择铅10μmol·L -1为铅暴露浓度。与正常对照组相比，单独铅或铜处理组 SOD 活性下降，MDA 含量增高，而铅与铜联合处理组 SOD 活性下降及 MDA 含量增高的程度更加显著（P ＜0．01）。与正常对照组比较，醋酸铅10μmol·L -1处理 C6细胞24和48 h 后，细胞对铜的吸收量分别增加1．2倍和2．5倍（P ＜0．01），CTR1 mRNA 水平分别增高23．2％和58．7％（P ＜0．01），而 ATP7A mRNA 水平分别下调58．1％和50．0％（P ＜0．01）。与正常对照组比较，醋酸铅10μmol·L -1暴露组 CTR1蛋白水平显著增高，而ATP7A 蛋白表达降低（P＜0．01）。结论铅暴露可以导致细胞中铜的蓄积和氧化应激反应的增强，其分子机制可能与影响 C6细胞 CTR1和 ATP7A 的表达水平有关。

OBJECTIVE To study the roles of copper transporter 1 （CTR1 ）and Cu2 ＋ transporting ATPase αpolypeptide （ATP7A）in lead exposure-induced copper accu mulation and oxidative stress in rat C6 glio ma cells.METHODS Methyl thiazolyl tetrazoliu m （MTT）assay was performed to determine the proper Pb doses （without affecting cell viability）by treated the cells with lead acetate 0 -100 μmol·L -1 for 24 h and 48 h.Superoxide dis mutase （SOD）activity or malondialdehyde（MDA）level were detected respectively by xanthine oxidase technique and thiobarbituric acid （TBA） method.Ato mic absorption spectrophoto metry was e mployed to determine the intracellular levels of Pb and Cu ions.Real-ti me quan-titative PCR and Western blot were used to detect the mRNA and protein levels of CTR1 and ATP7A, respectively.RESULTS The cell viability significantly decreased when the doses of Pb treat ment was higher than 10 μmol·L -1 ,so 10 μmol·L -1 was chosen as a working concentration of Pb exposure in this study.Co mpared with those in the normal controls,a moderately decreased T-SOD activity and an increased MDA level was determined in the cells treated with Pb 10 μmol·L -1 or Cu 5 μmol·L -1 alone, while a significant drop of T-SOD activity and a re markable increase of MDA level was found in the cells co-exposed to Pb and Cu （P〈0.01 ）.Pb exposure for 24 and 48 h increased the cellular Cu uptake by 1 .2 and 2.5 fold,respectively （P 〈0.01 ）.Evidences fro m RT-PCR showed that Pb exposure for 24 and 48 h upregulated the CTR1 mRNA level by 23.2% and 58.7%,and downregulated the ATP7A mRNA level by 58.1 % and 50.0%,respectively.Results fro m Western blot confirmed that Pb exposure also resulted in an increased CTR1 expression and a decreased ATP7A expression at protein level （P〈0.01 ）.CONCLUSION Pb exposure lead to Cu accu mulation,by affecting the expression levels of CTR1 and ATP7A,and increased oxidative stress in C6 cells.