6-羟基多巴胺致PC12细胞损伤机制中自噬的影响

Effects of an autophagy/lysosomal pathway induced by 6-hydroxydopamine in PC12 cells

目的 研究自噬在帕金森病（PD）细胞模型中的作用及可能的机制.方法 体外培养的PC12细胞加入6-羟基多巴（6-OHDA）诱导多巴胺能神经元损伤模型.利用透射电镜观察PC12细胞中自噬的激活,免疫印迹法检测LC3-Ⅱ、Cathepsin B蛋白的表达.结果 电镜下观察到6-OHDA可使PC12细胞内自噬体增多,并出现了凋亡特征.6-OHDA作用2h（灰度比：52.57±2.27）,4h（灰度比：56.83±3.51）,6h（灰度比：73.43±5.41）,12h（灰度比：103.90±2.57）,24h（灰度比：100.40±3.91）时LC3-Ⅱ表达逐渐升高,与正常对照组（42.10±2.05）比较差异有统计学意义（P＜0.05）,模型组Cathepsin B（113.80±4.46）表达与正常对照组（35.89±3.40）比较明显增加（P＜0.01）,与模型组相比,广谱蛋白酶抑制剂UTI组（57.69±4.24）降低Cathepsin B表达（P＜0.01）.结论 自噬/溶酶体途径参与PC12细胞的死亡过程：6-OHDA诱导自噬过度激活,LC3-Ⅱ与Cathepsin B表达增加,促进细胞死亡.

Objective To investigated the role of the autophagy lysosomal pathway in PD cells and the possible molecular mechanisms. Methods A dopaminergic neuronal injury model was induced by 6-OHDA in PC12 cells . Autophagosomes in PC12 cells were examined by transmission electronmicro-scopy（ TEM ）. The expression of LC3- Ⅱ , Cathepsin B were assayed by western blot analysis. Results TEM revealed that the autophagosomes were increased in PC12 cells after 6-OHDA treatment and appeared apoptosis. The LC3-Ⅱ （2h：52.57 ±2.27,4h：56.83 ±3.51,6h：73.43 ±5.41,12h：103.90 ±2.57,24h： 100.40 ±3.91 ）and Cathepsin B expression （ model group： 113.80 ± 4.46; normal group 35.89 ± 3.40） were increased after 6-OH DA treatments （P 〈 0.05 or P 〈 0.01 ）. Conclusion The results indicate that autophagy lysosome pathway is involved in 6-OHDA-induced cell death in PC12 cells.