Ctr1基因mRNA转录水平与细胞内铜离子浓度的相关性

Relationship of Ctr1 mRNA Transcription Level to Intracellular Copper Ion Concentration

目的分析铜特异性转运蛋白(Ctr1)基因mRNA转录水平与细胞内铜离子浓度的相关性。方法从猪传代肾细胞PK15中扩增Ctr1基因,克隆并测序;采用半定量RT-PCR法检测不同浓度铜离子(0、7.8、15.6、31.2和62.5μmol/L)对PK15细胞中Ctr1基因mRNA转录水平的影响,以β-actin为内参;采用原子吸收光谱分析法检测细胞内铜离子的浓度。结果所扩增的Ctr1基因与GenBank公布的序列同源性达到98%;PK15细胞Ctr1基因mRNA的转录水平在铜添加量在7.8～62.5μmol/L范围内呈双相反应,各试验组Ctr1基因转录水平均低于0μmol/L;而细胞内铜离子浓度均高于0μmol/L,以Ⅳ组含量最高,且与其他各组差异有统计学意义。结论 Ctr1基因mRNA的转录水平与细胞内铜离子浓度呈负相关。

Objective To analyze the relationship of transcription level of copper transport protein 1（Ctr1）mRNA to the copper ion concentration in cells.Methods Ctr1 gene was amplified from subcultured porcine kidney PK15 cells,then cloned and sequenced.The effects of various copper ion concentrations（0,7.8,15.6,31.2 and 62.5 μmol /L）on the Ctr1 mRNA transcription level in PK15 cells were determined by semi-quantitative RT-PCR,using β-actin as an internal reference.The intracellular copper ion concentration was determined by atomic absorption spectroscopy.Results The homology of amplified Ctr1 gene to that reported in GenBank was 98%.The transcription level of Ctr1 mRNA showed a double-way response to the copper ion concentration at a range of 7.8 ～ 62.5 μmol /L in PK15 cells.The Ctr1 mRNA transcription levels of various test groups were lower than that in control group（copper ion concentration 0 μmol /L）.However,the intracellular copper ion concentration in test groups were higher than that in control group,of which that in group Ⅳ（copper ion concentration 31.2 μmol /L）was significantly higher than those in the other groups.Conclusion The transcription level of Ctr1 mRNA was negatively related to intracellular copper ion concentration.