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目的探讨DJ-1对MN9D细胞的保护作用及具体机制。方法在MN9D细胞中瞬时转染DJ-1质粒,利用MTT法及流式细胞术分别检测MN9D细胞活力与凋亡水平。通过激光共聚焦显微镜(confocal)观察LC3自噬点数量。利用Western blot法检测LC3II/LC3I的比值。结果过表达DJ-1能部分恢复鱼藤酮引起的细胞活力下降(P<0.05)且可以减少鱼藤酮引起的MN9D细胞凋亡(P<0.05)。过表达DJ-1增加了鱼藤酮处理的MN9D细胞内荧光点的数量(P<0.001)且使LC3Ⅱ/Ⅰ的比值显著增高(P<0.001),应用3-MA干预后,细胞内荧光点数量回落到基础水平,且LC3Ⅱ/Ⅰ的比值明显降低(P<0.05)。同时应用自噬抑制剂3-MA干预后,DJ-1的细胞保护作用消失。结论 DJ-1通过激活自噬,起保护作用能减轻鱼藤酮引起的毒性。
Objective To investigate the protective effect and mechanism of DJ-1 on MN9D cells. Methods DJ-1 plasmid was transiently transfected into MN9D cells. The viability and apoptosis of MN9D cells were detected by MTT assay and flow cytometry respectively. The number of LC3 autophagy spots was observed by confocal microscopy. The ratio of LC3II / LC3I was determined by Western blot. Results DJ-1 overexpression partially restored rotenone-induced cell viability (P <0.05) and decreased rotenone-induced MN9D cell apoptosis (P <0.05). Overexpression of DJ-1 increased the number of fluorescent spots in MN9D cells treated with rotenone (P <0.001) and significantly increased the ratio of LC3II / I (P <0.001). After 3-MA intervention, the number of intracellular fluorescence spots dropped To the basal level, and the ratio of LC3Ⅱ / Ⅰ was significantly decreased (P <0.05). At the same time, the cytoprotective effect of DJ-1 disappeared after the intervention of autophagy inhibitor 3-MA. Conclusion DJ-1 can reduce the toxicity caused by rotenone by activating autophagy.