鱼藤酮对PC12细胞多巴胺相关转运体的影响

Effects of rotenone on proteins associated with dopamine transportation in PC12 cells

探讨鱼藤酮对PC12细胞多巴胺转运体(DAT)和突触囊泡单胺转运体2(VMAT2)的影响。我们将PC12细胞经不同浓度的鱼藤酮处理24h后,利用HE染色观察PC12细胞的形态学变化;利用免疫组织化学法观察不同浓度的鱼藤酮对PC12细胞酪氨酸羟化酶(TH),DAT和VMAT2蛋白表达的影响;利用RT-PCR法检测鱼藤酮对PC12细胞DAT和VMAT2基因表达的影响;通过生化实验检测Na+/K+-ATP酶活性。实验结果显示:鱼藤酮浓度为1.0mmol/L时,PC12细胞出现了明显的形态学改变,胞浆染色不均、胞体呈圆形、细胞核形状不规则、核浆比例增加;TH蛋白阳性细胞数量减少;DAT蛋白和基因表达增加;VMAT2蛋白和基因表达降低,与对照组比较均具有统计学意义(P<0.05),且随着鱼藤酮浓度的增加,变化趋势更为明显。鱼藤酮浓度为1.0mmol/L时,Na+/K+-ATP酶活力与对照组比较显著降低(P<0.05)。因此,DAT和VMAT2的表达异常可能参与了毒性作用。

To investigate the effects of rotenone on dopamine transporter （DAT） and vesicular monoamine transporter 2 （ VMAT2 ） in PC12 cells. PC12 cells were exposed to rotenone with different concentrations for 24 h. Then HE staining was used to observe the action of rotenone on the morphology of PC12 cells. The expression changes of TH, DAT and VMAT2 proteins in PC12 cells were observed with immunohistochemical staining. RT-PCR was employed to detect the mRNA expressions of DAT and VMAT2 in PC12 cells. Na ^＋/K^＋ -ATPase activity of the PC12 cells was measured biochemically. The results showed that the morphology of PC12 cells was affected by 1.0 mmol/L rotenone, and cytoblastema dyeing is uneven, circular cell body, cell nucleus anomalous and karyoplasm proportion increases. The number TH positive ceils decreased in the experimental group compared with control group （ P 〈 0.05 ）. Protein and mRNA expressions of DAT and VMAT2 significantly increased and decreased respectively in the PC12 cells administrated with rotenone, which presented a dose-dependent manner. The activity of Na^＋/K^＋ -ATPase decreased significantly in the PCI2 cells administrated with 1.0 mmol/L rotenone compared to the control group （ P 〈 0.05 ）. These results suggest that the abnormal expressions of DAT and VMAT2 may be involved in the action of rotenone induced PC12 cells toxicity.