帕金森病模型细胞中p53、增殖细胞核抗原表达与细胞增殖和凋亡

Expressions of p53 and proliferative cell nucleoantigen with neuronal proliferation and apoptosis in a cell model of Parkinson′s disease

目的:探讨帕金森病(PD)模型细胞中p53、增殖细胞核抗原(PCNA)表达与细胞增殖、凋亡相关性及病理变化特征。方法:以不同浓度的1-甲基-4-苯基-吡啶离子(MPP^+)诱导PC12细胞损伤,建立PD模型,PC12细胞分为空白组和MPP^+不同浓度组(0.5 mmol/L、1 mmol/L、2 mmol/L)共4组,采用MMT方法检测每组细胞生存率;DCFH-DA检测活性氧水平(ROS);吖啶橙和溴化乙锭(AO/EB)凋亡染色及酶联免疫吸附法(ELISA)测定单链DNA;Western blot检测各组p53和PCNA表达。结果:随MPP^+浓度增加,与对照组相比,PC12细胞生存率及该细胞凋亡呈剂量依赖性降低(P<0.05或P<0.01),ROS生成逐渐增加与剂量正相关(P<0.05或P<0.01);PCNA蛋白表达逐渐减少,其上游信号p53蛋白表达逐渐升高。结论:MPP^+诱导PC12细胞凋亡可能机制之一是通过p53介导PCNA降解实现的。

Objective:To investigate the pathological characteristics and correlation of expressions of p53 and proliferating cell nuclear antigen(PCNA)with neuronal proliferation and apoptosis in a cell model of Parkinson′s disease.Methods:The cell model of Parkinson′s disease was created by rat pheochromocytoma(PC12)cells with different concentrations of MPP^+(0,0.5,1,2 mmol/L).The MTT assay was performed to detect the cells viability,reactive osygen species(ROS)was detected by DCFH-DA assay.AO/EB staining and ELISA assay were done to confirm cells apoptosis and ssDNA respectively.Western blot was performed to detect the expressions of p53 and PCNA.Results:The measurements revealed a decrease in cell viability and a increase in apoptotic neurons and DNA fragmentation(P<0.05 or P<0.01)following the exposure of PC12 cells to MPP^+in a dose-dependent manner,and exposure to MPP^+induced a significant increase in ROS production(P<0.05 or P<0.01)in PC12 cells.The results also revealed that PCNA protein expression was downregulated in PC12 cells treated with MPP^+,whereas p53 expression was upregulated.Conclusion:One of the possible mechanisms of MPP^+-induced PC12 apoptosis is achieved by p53-mediated PCNA degradation.