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

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PCI2细胞;Westernn 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PCI2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100μmol／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100μmol／L时caspase的活性也到达最大值,而当MPP^＋浓度超过100μmol／L时,caspase的活性急剧下降;（4）用100μmol／L的MPP^＋处理PC12细胞24h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100μmol／LMPP^＋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。

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

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 PC12 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 PC12 cells. (2) MPP(+) caused a decrease of cell viability in a dose-dependent manner. At 100 mu mol/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 mu mol/mg protein) at 100 mu mol/L MPP(+). However caspase activity decreased significantly when the MPP(+) concentration exceeded 100 mu mol/L. (4) Overexpression of 14-3-3 protein decreased the apoptosis ratio of PC12 cells treated with 100 mu mol/L MPP(+) from 26.5% to 8.6%. (5) Bcl-2 protein tended to decrease but BAD protein tended to increase after treatment of PC12 cells with 100 mu mol/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.