6-羟多巴胺及囊泡单胺类转运功能抑制对PC12细胞凋亡的影响

Effect of 6-Hydroxydopamine and Inhibitors for Vesicular Monoamine Transporter on Apoptosis of PC12 Cells

目的探讨6-羟多巴胺(6-hydroxydopamine,6-OHDA)及囊泡单胺类转运体(vesicular monoamine transport-er,VMAT)功能抑制对大鼠嗜铬细胞瘤PC12细胞细胞凋亡的影响。方法不同浓度6-OHDA(25、50、100、200μmol/L)及VMAT功能抑制剂利血平(50、100、400、1 600nmol/L)与6-OHDA(100μmol/L)作用于PC12细胞,于不同时间点(12、24、36、48h)用流式细胞仪检测细胞凋亡率,并用Western blot法检测PC12细胞Bcl-2蛋白及Bax蛋白活性。结果加入不同浓度的6-OHDA时PC12细胞凋亡随6-OHDA浓度增加显著上升,并且引起Bcl-2蛋白表达抑制,Bax蛋白表达上升,具有时间依赖性。加入利血平后,相应的细胞凋亡增多,Bcl-2蛋白表达降低明显,Bax蛋白表达增多,差异有统计学意义。结论研究提示6-OHDA能诱导PC12细胞凋亡,并呈剂量及时间依赖性,其作用机制涉及到VMAT功能抑制触发了6-OHDA的内源性毒性,抑制Bcl-2蛋白的表达,促进细胞内Bax的表达,进而诱发多巴胺能神经元的凋亡。

Objective To study the effect of 6-Hydroxydopamine（6-OHDA）and inhibitors for vesicular monoamine transporter（VMAT）on apoptosis of PC12 cells.Methods Using flow cytometry,we detected the apoptosis of PC12 cells after exposure to different concentrations of 6-OHDA（25,50,100,200 μmol/L）,and VMAT inhibitors,reserpine（50,100,400,1 600 nmol/L）combined with 6-OHDA（100 μmol/L）for different durations（12,24,36,and 48 h）.Western blot was applied to measure the expression of Bcl-2 and Bax protein in PC12 cells.Results The apoptosis rate in PC12 cells was increased with the increase in 6-OHDA concentrations.Western blot revealed that the expression of Bcl-2 was reduced,whereas the expression of Bax was increased with the increase of 6-OHDA concentrations in a time-dependent manner.Combined use of reserpine and 6-OHDA could significantly increase the apoptosis rate and the expression of Bax,and decrease the expression of Bcl-2 in PC12 cells.Conclusion 6-OHDA can induce apoptosis of PC12 cells in a concentration-and time-dependent manner.The mechanisms may involve the neurotoxicity of 6-OHDA,and inhibitors for VMAT can aggravate the neurotoxicity through reducing the expression of Bcl-2 protein and enhancing the expression of Bax protein in PC12 cells.