中文摘要：

目的探讨铜转运蛋白和铜伴侣蛋白在醋酸铅染毒致大鼠神经胶质瘤细胞株C6细胞铜蓄积中的作用。方法 1采用CCK-8实验,以终浓度为0～50μmol/L醋酸铅处理C6细胞24.0 h,筛选合适的后续实验染毒剂量。2将C6细胞分为对照组和铅染毒组,分别予终浓度为0和10μmol/L醋酸铅处理24.0 h,再予浓度为2μmol/L的氯化铜培养,于培养后0.0、0.5、1.0、2.0、4.0和8.0 h收获细胞。以电感耦合等离子体-质谱法检测C6细胞内铜和铅水平,荧光实时定量聚合酶链式反应检测C6细胞中铜转运蛋白铜转运体1（CTR1）、二价金属转运体1（DMT1）、铜转运三磷酸腺苷酶α肽/β肽（ATP7A/ATP7B）和铜伴侣蛋白抗氧化蛋白1（ATOX1）、细胞色素C氧化酶17（COX17）、超氧化物歧化酶铜伴侣蛋白（CCS）的mRNA表达,以激光共聚焦检测铅染毒后细胞中CTR1和ATP7A的蛋白表达。结果 1CCK-8实验结果显示,浓度为10μmol/L的醋酸铅对C6细胞增殖尚未造成有统计学意义的影响（P〉0.05）,以该浓度作为后续铅染毒剂量。2醋酸铅染毒24.0 h后,铅染毒组C6细胞中铅和铜的水平均高于对照组（P〈0.01）,但2组C6细胞存活率比较,差异无统计学意义（P〉0.05）。经染铜处理后,铅染毒组C6细胞存活率低于对照组（P〈0.01）。C6细胞中铜水平在铅染毒处理和染铜时间的交互效应间有统计学意义（P〈0.01）;其中,在染铜后0.5～8.0 h 5个时间点,铅染毒组C6细胞中铜水平均高于对照组（P〈0.05）;对照组C6细胞中铜水平在染铜后2.0 h时间点达到高峰,此后直至8.0 h时间点均维持在较为平稳的水平;而铅染毒组C6细胞中铜水平呈随着染铜时间的增加而升高的时间-效应关系,在染铜后8.0 h时间点达到高峰。醋酸铅染毒24.0 h后,与对照组比较,铅染毒组C6细胞中CTR1和DMT1的mRNA相对表达水平分别上调113.00%和36.00%（P〈0.01）,ATP7A mRNA相对表达水平下调25.00%（P〈0.01）,CTR1?

英文摘要：

Objective To investigate the role of copper transporter protein and copper chaperones in copper accumulation in glioma cell line C6 cells induced by lead acetate exposure. Methods i） CCK-8 assay was used to determine the proper lead acetate dose by treating the cells with lead acetate at the final concentration of 0-50 μmol / L for 24. 0 hours. ii） C6 cells were divided into control group and lead-exposure group,treated with 0 and 10 μmol / L lead acetate respectively for24. 0 hours,and then cultured in 2 μmol / L copper chloride for 0. 0,0. 5,1. 0,2. 0,4. 0 and 8. 0 hours; inductively coupled plasma mass spectrometry was used to detect the levels of copper and lead in the cells. Real-time polymerase chain reaction was used to detect the mRNA expression of copper transporter 1（ CTR1）,divalent metal transporter 1（ DMT1）,copper-transporting ATPase α polypeptide / β polypeptide（ ATP7 A and ATP7B）, antioxidant 1 copper chaperone（ ATOX1）,cytochrome c oxidase copper chaperone（ COX17）,and copper-chaperone-for-superoxide dismutase（ CCS）.Laser con-focal microscopy was applied to detect the protein expression of CTR1 and ATP7 A in cells. Results i） CCK-8assay proved that the 10 μmol / L lead acetate treatment did not affect C6 cells proliferation（ P〈0. 05）. Thus the final concentration of 10 μmol / L lead acetate was chosen as the treatment dose in later experiments. ii） After 10 μmol / L lead acetate exposure for 24. 0 hours,the lead and copper levels of C6 cells in lead-exposure group were higher than those in the control group（ P〈0. 01）,but there was no statistical significant difference in the C6 cell survival rate between these two groups（ P〈0. 05）. After cells were treated with copper,the C6 cell survival rate of lead-exposure group was lower than that in the control group（ P〈0. 01）. The interactive effect of copper level showed statistical significance between lead exposure and cooper treatment time（ P〈0. 01）. At the 5 time points from 0. 5-8. 0 h