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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。
Objective To investigate the effect of 14-3-3 protein overexpression on the death of PC12 cells induced by 1-methyl-4-phenylpyridinium ion (MPP +) and its possible mechanism. Methods The pcDNA3.1 (+) - 14-3-3 eukaryotic expression plasmid was constructed and transfected into PC12 cells by lipofectamine 2000. The expression of 14-3-3 protein, Bcl-2 protein and BAD protein in PC12 cells were detected by Western blot. Then the activity of PC12 cells, the activity of caspase and the apoptosis rate of PC12 cells were detected by MTT assay, microplate reader and flow cytometry respectively. Results (1) After transfected pcDNA3.1 (+) - 14-3-3 plasmid into PC12 cells for 3 weeks, the expression of 14-3-3 protein was significantly increased. (2) The decrease of survival rate of PC12 cells induced by MPP + Dependently, the viability of PC12 cells was lost about 50% when the MPP + concentration was 100 μmol / L; (3) the activity of caspase increased with the increase of MPP + concentration. When MPP + concentration reached 100 μmol / L, caspase The activity of caspase decreased sharply when the concentration of MPP + exceeded 100 μmol / L. (4) The apoptosis rate of PC12 cells treated with 100 μmol / L MPP + for 24 h was 26.5% , 14-3-3 protein overexpression decreased the apoptosis rate of PC12 cells to 8.6%. (5) After treatment of PC12 cells with 100 μmol / L MPP +, the expression of Bcl-2 protein tended to decrease and the expression of BAD protein Upregulation, 14-3-3 protein overexpression can significantly increase the expression of Bcl-2 protein and BAD protein expression down. Conclusion Overexpression of 14-3-3 can reduce the apoptosis of PC12 cells induced by MPP + and up-regulate the expression of Bcl-2 protein and down-regulate the expression of BAD protein, thereby protecting PC12 cells. These results may provide new drug targets for the treatment of PD.