Overexpression of 14-3-3 protein protects pheochromocytoma cells against 1-methyl-4-phenylpyridinium toxicity  

14-3-3蛋白过表达减轻1-甲基-4-苯基吡啶离子对PC12细胞的毒性

作　　者：陈小武[1] 孙圣刚[1] 称道宾[1] 田有勇[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院神经内科,武汉430022

出　　处：《Neuroscience Bulletin》2006年第5期281-287,共7页神经科学通报（英文版）

基　　金：supported by National Natural Science Foundation of China(No:30570627).

摘　　要：Objective To investigate the effects of 14-3-3 protein overexpression on the 1-methyl-4-phenylpyridinium （MPP^＋） induced pheochromocytoma （PC12） cell death and the potential mechanisms. Methods pcDNA3.1（＋）-14-3-3 plasmids, which could be expressed in mammalian cell, were constructed and transfected into PC 12 cells with Lipofectamine 2000. The expression of 14-3-3 protein, Bcl-2 protein, and BAD protein were determined by western blot. 3-（4,5- dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay, microplate reader, and flow cytometric analysis were used to measure cell viability, the caspase activity, and apoptotic ratio respectively. Results （1） The expression of 14-3-3 protein increased significantly three weeks after pcDNA3.1（＋）-14-3-3 plasmids transfected into PC 12 cells. （2） MPP^＋ caused a decrease of cell viability in a dose-dependent manner. At 100μmol/L MPP^＋, cell viability reduced approximately 50%. （3） The caspase activity increased along with the MPP^＋ concentrations rising and reached its maximum value （0.34 μmol/mg protein） at 100 μmol/L MPP＊. However caspase activity decreased significantly when the MPP^＋ concentration exceeded 100 μmol/L. （4） Overexpression of 14-3-3 protein decreased the apoptosis ratio of PC 12 cells treated with 100μmol/L MPP^＋ from 26.5% to 8.6%. （5） Bcl-2 protein tended to decrease but BAD protein tended to increase after treatment of PC 12 cells with 100 μmol/L MPP^＋. Overexpression of 14-3-3 protein significantly increased the cellular level of Bcl-2 protein and decreased that of BAD protein. Conclusion Overexpression of 14-3-3 protein may reduce MPP^＋-induced apoptotic cell death in PC12 cells by up-regulating the Bcl-2 expression and down-regulating the BAD expression. These results may provide a promising target for treatment of Parkinson＇s disease.目的研究14-3-3蛋白过表达对1-甲基-4苯基吡啶离子(MPP+)诱导的PC12细胞死亡的影响作用及其可能的机制。方法构建pcDNA3.1(+)-14-3-3 真核表达质粒,用脂质体2000转染PC12细胞;Western blot技术检测PC12细胞中14-3-3蛋白、Bcl-2 蛋白, 和BAD蛋白的表达;然后分别用MTT法、酶标仪及流式细胞仪检测PC12细胞的活力、caspase的活性及PC12细胞的凋亡率。结果(1)将pcDNA3.1(+)-14-3-3质粒转染PC12细胞3周后,14-3-3蛋白的表达显著增加;(2)MPP+诱导PC12细胞存活率的下降是剂量依赖性的,当MPP+的浓度达100 μmol/L时,PC12细胞的存活率丧失约50%;(3)caspase 的活性随着MPP+浓度的增加而增高,当MPP+浓度到达100 μmol/L时caspase的活性也到达最大值,而当MPP+浓度超过100 μmol/L时,caspase的活性急剧下降;(4)用100 μmol/L 的MPP+处理PC12细胞24 h后,PC12细胞的凋亡率为26.5%,14-3-3蛋白的过表达使PC12细胞的凋亡率下降到8.6%;(5)用100 μmol/L MPP+处理PC12细胞后,Bcl-2蛋白的表达趋于下调而BAD蛋白的表达上调,14-3-3蛋白的过表达能显著的增加Bcl-2蛋白的表达而使BAD蛋白的表达下调。结论14-3-3蛋白过表达通过上调Bcl-2蛋白的表达并下调BAD蛋白的表达,减少了MPP+诱导的PC12细胞的凋亡,从而发挥对PC12细胞的保护作用。这些结果可能为PD的治疗提供新的药物靶点。

关 键 词：14-3-3 protein MPP^＋ PC 12 cell apoptosis Parkinson＇s disease 

分 类 号：R742.5[医药卫生—神经病学与精神病学]