锌对原代培养海马神经元锌转运体1和金属硫蛋白mRNA表达的影响

Effect of zinc on expression of zinc transporter 1 and metallothionein mRNA in primary cultured hippocampal neurons

目的 :观察不同浓度锌对锌转运体 1 (zinc transporter1 ,Zn T- 1 ) m RNA和金属硫蛋白 (metallothionein,MT) 1、2m RNA表达的影响 ,旨在探讨神经元锌内稳态的可能机制。 方法 :采用锥虫蓝染色法检测不同浓度锌 (0、5 0、1 0 0、1 2 5、1 5 0、1 75、2 0 0 μm ol/ L)对原代培养海马神经元存活率的影响 ;用实时荧光定量 RT- PCR法分别检测不同浓度锌 (0、5 0、75、1 0 0、1 2 5、1 5 0 μmol/ L)对 Zn T- 1、MT1、MT2的 m RNA表达的影响 ,检测 1 0 0 μm ol/ L 锌暴露不同时间点 (0、2、4、6、8h)对 Zn T- 1、MT1、MT2的 m RNA表达的影响。 结果 :培养基锌浓度大于 1 2 5 μmol/ L 时 ,海马神经元存活率明显下降 ;锌浓度大于 75μm ol/ L 时 ,Zn T- 1 m RNA表达成线性上升 (P<0 .0 5 ) ,MT m RNA表达同样明显上升 (P<0 .0 5 ) ,但在 1 0 0 μm ol/ L 后进入平台期 ;在 1 0 0 μm ol/ L 锌浓度条件下 ,Zn T- 1 m RNA表达在 2 h内达到峰值 ,MT m RNA在 6 h达到其峰值。结论 :虽然神经元可通过增加 Zn T- 1和 MT的表达来应对高锌冲击 ,但两者的保护作用不同 ,Zn T- 1外排作用发生在前且具有持续性 。

Objective:To study the concentration response and time course of zinc transporter 1 (ZnT 1) and metallothionein (MT1/MT2) mRNA,as well as the cell viability to zinc exposure in primary cultured new born rats hippocampal neuron s. Methods: The cell viability were determined by trypan blue s taining at various concentrations of Zn 2+ (0, 50, 100, 125, 150, 175, 200 μmol/L). The expression of ZnT 1, MT1 and MT2 mRNA to various concentrations of zinc exposure(0, 50, 75, 100, 125, 150 μmol/L) and to 100 μmol/L zinc exposure for different times (0, 2, 4, 6, 8 h) were determined b y real time fluorescent quantitative PCR. Results: The viabilit y of the neurons decreased significantly after 48 h once the concentration of Zn 2+ exceeded 125 μmol/L,and the expression of ZnT 1 mRNA was in proportion to the increment of zinc.The expression of MT1/MT2 mRNA reached a plateau when the zinc concentration exceeding 75 μmol/L. The expression of ZnT 1 mRNA peaked on about 2 h.However, the expression of MT1/MT2 mRNA reached its maximal around 6 h at the concentration of 100 μmol/L. Conclusion: These results imply that although both MTs and ZnT 1 can attenuate the zinc toxicity, they may play different roles at different phases. ZnT 1 enhance the efflux of zinc prior to the sequesteration by MTs. Th e action of ZnT 1 may be durable, but the role of MTs may be satiable.