14-3-3蛋白过表达减轻MPP^+对PC12细胞的毒性损伤

Overexpression of 14-3-3 protein alleviates the toxicity of MPP^+ to PC12 cell

目的探讨14-3-3蛋白过表达对1-甲基-4-苯基吡啶离子(MPP^+)诱导 PC12细胞损伤的保护作用和可能的机制。方法构建 pcDNA3.1(+)-14-3-3真核表达质粒,转染 PC12细胞,建立稳定过表达14-3-3蛋白细胞株;通过四甲基偶氮唑盐法(MTT 法)、流式细胞术和酶标仪分别检测14-3-3蛋白过表达对 MPP^+诱导的 PC12细胞存活力、凋亡率和 SOD 及谷胱甘肽过氧化物酶(GSH-Px)活性的影响。结果 14-3-3蛋白过表达显著增加 PC12细胞 SOD 活性[质粒转染组(9.13±0.41)U/mg,MPP^+组(6.45±0.52)U/mg]和 GSH-Px 活性[质粒转染组(89.66±3.42)μmol/mg,MPP^+组(82.73±4.15)μmol/mg]、增强细胞活力[吸光度(A_(570)):质粒转染组0.78±0.06,MPP^+组0.54±0.07]、抑制细胞的凋亡(质粒转染组11.87%±3.26%,MPP^+组36.30%±2.39%)。结论 14-3-3蛋白过表达对 MPP^+的毒性有保护作用,这是通过增加 SOD 和 GSH-Px 的活性,减少氧化应激实现的。

Objective To investigate the protective effects of the 14-3-3 protein overexpression on the injury of PC12 cell induced by MPP^＋ and its mechanisms. Methods For expression in mammalial cells, pcDNA3. 1 （±）-14-3-3 plasmid was constructed and transfected into PC12 cell with Lipofectamine^TM 2000. The overexpression of transfected 14-3-3 gene in PC12 cell was determined by immunofluorescence and Western blotting. The effects of 14-3-3 overexpressing on the cells viability, apoptotic ratio and the activity of superoxide dismutase （SOD） as well as glutathione peroxidase （GSH-Px） of PC12 cell treated with MPP^＋ were measured by MTT assay, flow cytometry analysis and microplate reader respectively. Results The expression of 14-3-3 protein in transfection group （1.19 ± 0. 06） increased evidently compared with control group （0. 75 ± 0. 05 ）. And the antioxidant enzyme activity assession , MTT assay and flow cytometry analysis shows that the overexpression of 14-3-3 protein elevates the activity of SOD （transfection group： （9.13 ± 0.41） U/mg protein, MPP^＋ group： （6.45 ± 0.52 ） U/mg protein） and GSH-Px （transfection group： （ 89. 66 ± 3.42 ） μmol/mg , protein MPP^＋ group： （ 82.73 ± 4. 15 ） μmol/mg protein ）, increases the cell viability （transfection group： 0. 78 ± 0. 06, MPP^＋ group： 0. 54 ± 0. 07 ）, and inhibits cell apoptosis （transfection group： 11.87% ±3.26% ,MPP^＋ group：36.30% ±2.39%） of PC12 induced by MPP^＋. Conclusion The overexpression of 14-3-3 protein could elevate the activity of antioxidant enzymes SOD and GSH-Px, reduce oxidant stress, alleviate MPP^＋ toxicity , and thus inhibit the apoptosis of PC12 cell induced by MPP^＋.