铜转运相关蛋白在醋酸铅致C6细胞铜蓄积中作用
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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 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 hours after exposure to copper,the copper levels in lead-exposure group were higher than those of control group( P < 0. 05). The copper levels in the control group reached a peak after exposure to copper for 2. 0 hours,and maintained at a stable level till the time point of 8. 0hours. The copper levels of lead-exposed groups increased with the increasing time of copper exposure and there was a time-effect relationship,and they reached to the peak at the time point of 8. 0 hours. After 10 μmol / L lead acetate exposure for 24. 0 hours,compared with control group,the CTR1 and DMT1 mRNA relative expression levels in leadexposed group increased by 113. 00% and 36. 00% respectively( P < 0. 01),and the ATP7 A mRNA relative expression level decreased by 25. 00%( P < 0. 01). The protein expression of CTR1 increased by 76. 04%( P < 0. 01),and the protein expression of ATP7 A decreased by 16. 0%( P < 0. 01). There was no significant difference in the mRNA relative expression levels of ATP7 B,ATOX1,COX17 and CCS between the two groups( P > 0. 05). Conclusion Lead acetate exposure can lead to increase accumulation of copper in C6 cells with increasing exposure time showing a time-effect relationship. The increased protein expression of CTR1 and decreased protein expression of ATP7 A might be one of the mechanisms of inducing copper accumulation in cells after the lead acetate exposure.
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 hours after exposure to copper,the copper levels in lead-exposure group were higher than those of control group( P < 0. 05). The copper levels in the control group reached a peak after exposure to copper for 2. 0 hours,and maintained at a stable level till the time point of 8. 0hours. The copper levels of lead-exposed groups increased with the increasing time of copper exposure and there was a time-effect relationship,and they reached to the peak at the time point of 8. 0 hours. After 10 μmol / L lead acetate exposure for 24. 0 hours,compared with control group,the CTR1 and DMT1 mRNA relative expression levels in leadexposed group increased by 113. 00% and 36. 00% respectively( P < 0. 01),and the ATP7 A mRNA relative expression level decreased by 25. 00%( P < 0. 01). The protein expression of CTR1 increased by 76. 04%( P < 0. 01),and the protein expression of ATP7 A decreased by 16. 0%( P < 0. 01). There was no significant difference in the mRNA relative expression levels of ATP7 B,ATOX1,COX17 and CCS between the two groups( P > 0. 05). Conclusion Lead acetate exposure can lead to increase accumulation of copper in C6 cells with increasing exposure time showing a time-effect relationship. The increased protein expression of CTR1 and decreased protein expression of ATP7 A might be one of the mechanisms of inducing copper accumulation in cells after the lead acetate exposure.
引文
[1]MENG H,WANG L,HE J,et al.The protective effect of gangliosides on lead(Pb)-induced neurotoxicity is mediated by autophagic pathways[J].Int J Environ Res Public Health,2016,13(4).doi:10.3390/ijerph13040365.
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[8]郑创亮,许丹,陈青松,等.工作场所健康促进和职业安全健康管理体系对某铅蓄电池企业铅污染干预效果分析[J].中国职业医学,2014,41(3):315-318.
[9]谢春姣,刘汝青,袁惟芯,等.DNA甲基化转移酶1基因多态性与铅中毒易感性分析[J].中国职业医学,2014,41(2):121-125.
[10]WANG T,GUAN R L,LIU M C,et al.Lead exposure impairs hippocampus related learning and memory by altering synaptic plasticity and morphology during juvenile period[J].Mol Neurobiol,2015:1-13.
[11]张洁琼,于海波,宋晗,等.铅暴露对神经胶质瘤细胞铜代谢的影响及其机制[J].中国药理学与毒理学杂志,2014,28(2):182-187.
[12]MARCHETTI C,BARANOWSKA-BOSIACKA I,Gavazzo P.Multiple effects of copper on NMDA receptor currents[J].Brain Res,2014,1542:20-31.
[13]SALAZAR-WEBER N L,SMITH J P.Copper inhibits NMDA receptor-independent LTP and modulates the paired-pulse ratio after LTP in mouse hippocampal slices[J].Int J Alzheimers Dis,2011:864753.doi:10.4061/2011/864753.
[14]LEIVA J,PALESTINI M,INFANTE C,et al.Copper suppresses hippocampus LTP in the rat,but does not alter learning or memory in the morris water maze[J].Brain Res,2009,1256:69-75.
[15]ZHOU G,JI X,CUI N,et al.Association between serum copperstatus and working memory in schoolchildren[J].Nutrients,2015,7(9):7185-7196.
[16]YU J,LUO X,XU H,et al.Identification of the key moleculesinvolved in chronic copper exposure-aggravated memory impairment in transgenic mice of Alzheimer's disease using proteomic analysis[J].J Alzheimers Dis,2015,44(2):455-469.
[17]赵会新,杨辉,闫立成,等.染铅大鼠脉络丛中铜及铜转运蛋白的变化[J].中华劳动卫生职业病杂志,2014,32(11):819-822.
[18]PUSHIE M J,SHAW K,FRANZ K J,et al.Model peptide studies reveal a mixed histidine-methionine Cu(I)binding site at the N-terminus of human copper transporter 1[J].Inorg Chem,2015,54(17):8544-8451.
[19]BULCKE F,DRINGEN R.Handling of copper and copper oxide nanoparticles by astrocytes[J].Neurochem Res,2016,41(1/2):33-43.
[20]张洁琼.CTR1和ATP7A参与铅诱导血-脑脊液屏障铜蓄积的机制研究[D].西安:第四军医大学,2014.
[21]MATSON DZEBO M,ARIOZ C,WITTUNG-STAFSHEDE P.Extended functional repertoire for human copper chaperones[J].Biomol Concepts,2016,7(1):29-39.
[22]FU X,ZHANG Y,JIANG W,et al.Regulation of copper transport crossing brain barrier systems by Cu-ATPases:effect of manganese exposure[J].Toxicol Sci,2014,139(2):432-451.
[23]王超曼,程楠,韩咏竹,等.细胞内铜转运系统的研究进展[J].安徽卫生职业技术学院学报,2013,12(3):78-81.
[24]QIAN Y,ZHENG Y,RAMOS K S,et al.The involvement of copper transporter in lead-induced oxidative stress in astroglia[J].Neurochem Res,2005,30(4):429-438.
[2]LEE J,FREEMAN J L.Embryonic exposure to 10μg L-1 lead results in female-specific expression changes in genes associated with nervous system development and function and Alzheimer's disease in aged adult zebrafish brain[J].Metallomics,2016.doi:10.1039/C5MT00267B.
[3]WEUVE J,PRESS D Z,GRODSTEIN F,et al.Cumulative exposure to lead and cognition in persons with Parkinson's disease[J].Mov Disord,2013,28(2):176-182.
[4]SQUITTI R,SIOTTO M,POLIMANTI R.Low-copper diet as a preventive strategy for Alzheimer's disease[J].Neurobiol Aging,2014,35(Suppl 2):S40-50.
[5]ROBERT A,LIU Y,NGUYEN M,et al.Regulation of copper and iron homeostasis by metal chelators:a possible chemotherapy for Alzheimer's disease[J].Acc Chem Res,2015,48(5):1332-1339.
[6]DREW S C,NOBLE C J,MASTERS C L,et al.Pleomorphic copper coordination by Alzheimer's disease amyloid-beta peptide[J].J Am Chem Soc,2009,131(3):1195-1207.
[7]LONNERDAL B.Intestinal regulation of copper homeostasis:a developmental perspective[J].Am J Clin Nutr,2008,88(3):846S-850S.
[8]郑创亮,许丹,陈青松,等.工作场所健康促进和职业安全健康管理体系对某铅蓄电池企业铅污染干预效果分析[J].中国职业医学,2014,41(3):315-318.
[9]谢春姣,刘汝青,袁惟芯,等.DNA甲基化转移酶1基因多态性与铅中毒易感性分析[J].中国职业医学,2014,41(2):121-125.
[10]WANG T,GUAN R L,LIU M C,et al.Lead exposure impairs hippocampus related learning and memory by altering synaptic plasticity and morphology during juvenile period[J].Mol Neurobiol,2015:1-13.
[11]张洁琼,于海波,宋晗,等.铅暴露对神经胶质瘤细胞铜代谢的影响及其机制[J].中国药理学与毒理学杂志,2014,28(2):182-187.
[12]MARCHETTI C,BARANOWSKA-BOSIACKA I,Gavazzo P.Multiple effects of copper on NMDA receptor currents[J].Brain Res,2014,1542:20-31.
[13]SALAZAR-WEBER N L,SMITH J P.Copper inhibits NMDA receptor-independent LTP and modulates the paired-pulse ratio after LTP in mouse hippocampal slices[J].Int J Alzheimers Dis,2011:864753.doi:10.4061/2011/864753.
[14]LEIVA J,PALESTINI M,INFANTE C,et al.Copper suppresses hippocampus LTP in the rat,but does not alter learning or memory in the morris water maze[J].Brain Res,2009,1256:69-75.
[15]ZHOU G,JI X,CUI N,et al.Association between serum copperstatus and working memory in schoolchildren[J].Nutrients,2015,7(9):7185-7196.
[16]YU J,LUO X,XU H,et al.Identification of the key moleculesinvolved in chronic copper exposure-aggravated memory impairment in transgenic mice of Alzheimer's disease using proteomic analysis[J].J Alzheimers Dis,2015,44(2):455-469.
[17]赵会新,杨辉,闫立成,等.染铅大鼠脉络丛中铜及铜转运蛋白的变化[J].中华劳动卫生职业病杂志,2014,32(11):819-822.
[18]PUSHIE M J,SHAW K,FRANZ K J,et al.Model peptide studies reveal a mixed histidine-methionine Cu(I)binding site at the N-terminus of human copper transporter 1[J].Inorg Chem,2015,54(17):8544-8451.
[19]BULCKE F,DRINGEN R.Handling of copper and copper oxide nanoparticles by astrocytes[J].Neurochem Res,2016,41(1/2):33-43.
[20]张洁琼.CTR1和ATP7A参与铅诱导血-脑脊液屏障铜蓄积的机制研究[D].西安:第四军医大学,2014.
[21]MATSON DZEBO M,ARIOZ C,WITTUNG-STAFSHEDE P.Extended functional repertoire for human copper chaperones[J].Biomol Concepts,2016,7(1):29-39.
[22]FU X,ZHANG Y,JIANG W,et al.Regulation of copper transport crossing brain barrier systems by Cu-ATPases:effect of manganese exposure[J].Toxicol Sci,2014,139(2):432-451.
[23]王超曼,程楠,韩咏竹,等.细胞内铜转运系统的研究进展[J].安徽卫生职业技术学院学报,2013,12(3):78-81.
[24]QIAN Y,ZHENG Y,RAMOS K S,et al.The involvement of copper transporter in lead-induced oxidative stress in astroglia[J].Neurochem Res,2005,30(4):429-438.