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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蛋白表达水平上调76.04%(P<0.01),ATP7A蛋白表达水平下调16.00%(P<0.01)。2组C6细胞中ATP7B、ATOX1、COX17和CCS的mRNA相对表达水平分别比较,差异均无统计学意义(P>0.05)。结论醋酸铅染毒可导致C6细胞中铜水平呈随时间增加而增加的时间-效应性蓄积;铜转入蛋白CTR1表达的上调和铜转出蛋白ATP7A表达的下调可能是醋酸铅染毒后致细胞中铜蓄积的机制之一。
Objective To investigate the role of copper transporter and copper chaperone in the accumulation of copper in rat glioma C6 cell line induced by lead acetate. Method 1 CCK-8 was used to treat C6 cells at a final concentration of 0-50 μmol / L for 24 h, and the suitable dose of follow-up experiment was screened. The C6 cells were divided into the control group and the lead-exposed group, treated with 0 and 10 μmol / L lead acetate for 24 h, respectively, and then treated with 2 μmol / L cupric chloride. After culture, 0.0, 0.5, 1.0 Cells were harvested at 2.0, 4.0 and 8.0 h. The levels of copper and lead in C6 cells were detected by inductively coupled plasma-mass spectrometry. The expressions of copper transporter copper transporter 1 (CTR1), divalent metal transporter 1 (DMT1) The mRNA expression of copper transporters ATP7A / ATP7B and ATOX1, COX17 and CCS The protein expression of CTR1 and ATP7A in the cells was detected by laser confocal laser scanning microscope. Results 1CCK-8 experimental results showed that the concentration of 10μmol / L of lead acetate on C6 cell proliferation has not caused a statistically significant effect (P> 0.05), the concentration as the follow-up lead poisoning dose. The levels of lead and copper in C6 cells of lead-exposed group were higher than those in control group (P <0.01) after 24.0 h exposure to lead acetate, but there was no significant difference in the survival rate of C6 cells between two groups (P> 0.05) . After being treated with copper, the survival rate of C6 cells in lead-exposed group was lower than that in control group (P <0.01). The level of copper in C6 cells was statistically significant (P <0.01) between the lead exposure and the copper exposure time (P <0.01). Among the C6 cells in the lead-exposed group (P <0.05). In the control group, the level of copper in C6 cells reached the peak at 2.0 h after the copper dying, and remained stable at 8.0 h. The level of copper in C6 cells showed a time-effect relationship with the increase of copper exposure time, reaching the peak at 8.0 h after copper exposure. Compared with the control group, the mRNA relative expression of CTR1 and DMT1 in lead-exposed group was increased by 113.00% and 36.00% (P <0.01), and the relative expression level of ATP7A mRNA was decreased by 25.00% (P <0.01). The CTR1 protein level was up-regulated by 76.04% (P <0.01) and the ATP7A protein level by 16.00% (P <0.01). There was no significant difference in the mRNA relative expression levels of ATP7B, ATOX1, COX17 and CCS between C6 and C6 cells (P> 0.05). Conclusion Lead acetate can induce the time-dependent accumulation of copper in C6 cells with the increase of time. The up-regulation of CTR1 expression and the down-regulation of copper transfer protein ATP7A may be induced by lead acetate One of the mechanisms of copper accumulation.