联合6-羟多巴胺及囊泡单胺类转运功能抑制对PC12细胞凋亡的影响及机制

The 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-OHDA)及囊泡单胺类转运体(VMAT)功能抑制对大鼠嗜铬细胞瘤PC12细胞凋亡的影响、线粒体膜电位及细胞内Ca2+离子的改变。方法阴性对照、6-OHDA(25、50、100、200μmol/L)、VMAT功能抑制剂利血平(50、100、400、1 600 nmol/L)与6-OHDA(100μmol/L)作用于PC12细胞,前两大组于不同时间点(0、12、24、36、48 h),后一大组于0、24 h用流式细胞仪检测细胞凋亡率,并用JC-1染色流式细胞仪定量测定线粒体膜电位(△ψm)变化,激光共聚焦显微镜通过荧光探针强度检测PC12细胞内Ca2+离子浓度。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,具有时间依赖性(P<0.001)。PC12细胞△ψm降低比例随6-OHDA浓度增加而增加,并随作用时间的延长增加更加明显(P<0.001);细胞内Ca2+离子浓度随6-OHDA浓度增加而升高,并随作用时间的延长浓度升高更加明显。加入利血平后,相应的细胞凋亡增多,细胞内△ψm降低比例明显增加,Ca2+离子浓度升高,差异有统计学意义(P<0.001)。结论 6-OHDA能诱导PC12细胞凋亡,并呈剂量及时间依赖性;其作用机制可能为诱导PC12细胞内△ψm的降低及钙离子超载,并呈剂量及时间依赖性;VMAT功能抑制进一步加重了6-OHDA的内源性毒性,引起△ψm的降低及钙离子超载加重,进一步加重细胞凋亡。

Objective To study the effect of 6-Hydroxydopamine （6-OHDA） and inhibitors for vesicular monoamine transporter （VMAT） on mitochondrial membrane potential（△ψm） and intracellular free calcium concentration（[Ca2＋]i） in PC12 cells to investigate the mechanism of 6-OHDA induced apoptosis in PC12 cells.Methods The apoptosis of PC12 cells was detected by flow cytometry.The △Ψm was assayed by flow cytometry with JC-1 fluorescence staining.The level of [Ca-2＋]in PC12 cells was detected with fluorescent intensity（FI） by laser scanning confocal microscopy.The PC cells were divided into negative control,different concentrations of 6-OHDA （25,50,100,200 μ.mol/L） at different time（0,12,24,36,48 h） or exposured to VMAT inhibitors,reserpine （50,100,400,1 600 nmol/L）with 6-OHDA （100 μmol/L） at 0,24 h.Results The apoptotic rate in PC12 cell was increased with the 6-OHDA density elevating; The percentage of cells with reduction of △Ψm in PC12 cells was increased with the concentration elevating of 6-OHDA; The level of [Ca2＋]i in PC12 cells was increased with the concentration of 6-OHDA increasing,all the changes had the time dependence.Treatment with reserpine,the apoptotic rate in PC12 cell was increased in responding group,the percentage of cells with reduction of △Ψm in PC12 cells was increased and the level of [Ca-2＋]i in PC12 cells was increased in responding group,and there were significantly statistical differences.Conclusions 6-OHDA could induce PC12 cell apoptosis,and the effect has density and time dependence.The mechanisms might be inducing the reduction of △Ψm and increasing the level of [Ca2＋]i of PC12 cells,and the effect has concentration and time dependence.The inhibitors of VMAT could aggravate the neurotoxicitv.