Department of Neurology, the Affiliated Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China; E-mail:
Neurosci Bull. 2006 Sep;22(5):281-7.
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.
目的 探讨 14-3-3 蛋白过表达对 1-甲基-4-苯基吡啶（MPP(+））诱导的嗜铬细胞瘤（PC12）细胞死亡的影响及其潜在机制。
方法 构建可在哺乳动物细胞中表达的 pcDNA3.1(+)-14-3-3 质粒，并用脂质体 2000 将其转染至 PC12 细胞中。采用 Western blot 法检测 14-3-3 蛋白、Bcl-2 蛋白和 BAD 蛋白的表达。采用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）比色法、微孔板读数仪和流式细胞术分别测定细胞活力、半胱天冬氨酸蛋白酶（caspase）活性和凋亡率。
结果 （1）pcDNA3.1(+)-14-3-3 质粒转染 PC12 细胞 3 周后，14-3-3 蛋白表达明显增加。（2）MPP(+)呈剂量依赖性降低细胞活力，100 μmol/L MPP(+)时，细胞活力约降低 50%。（3）随着 MPP(+)浓度的升高，caspase 活性逐渐升高，在 100 μmol/L MPP(+)时达到最大值（0.34 μmol/mg 蛋白），但当 MPP(+)浓度超过 100 μmol/L 时，caspase 活性显著下降。（4）过表达 14-3-3 蛋白可使 100 μmol/L MPP(+)处理的 PC12 细胞的凋亡率从 26.5%降至 8.6%。（5）MPP(+)处理 PC12 细胞后，Bcl-2 蛋白呈下降趋势，BAD 蛋白呈上升趋势，而过表达 14-3-3 蛋白可显著增加细胞内 Bcl-2 蛋白水平，降低 BAD 蛋白水平。
结论 过表达 14-3-3 蛋白可能通过上调 Bcl-2 表达和下调 BAD 表达，减少 MPP(+）诱导的 PC12 细胞凋亡性死亡。这些结果可能为帕金森病的治疗提供一个有希望的靶点。
