CAJ | 학술논문

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.
목적 연구자서재파금삼병(PD)세포모형중적작용급가능적궤제.방법 체외배양적PC12세포가입6-간기다파(6-OHDA)유도다파알능신경원손상모형.이용투사전경관찰PC12세포중자서적격활,면역인적법검측LC3-Ⅱ、Cathepsin B단백적표체.결과 전경하관찰도6-OHDA가사PC12세포내자서체증다,병출현료조망특정.6-OHDA작용2h(회도비:52.57±2.27),4h(회도비:56.83±3.51),6h(회도비:73.43±5.41),12h(회도비:103.90±2.57),24h(회도비:100.40±3.91)시LC3-Ⅱ표체축점승고,여정상대조조(42.10±2.05)비교차이유통계학의의(P＜0.05),모형조Cathepsin B(113.80±4.46)표체여정상대조조(35.89±3.40)비교명현증가(P＜0.01),여모형조상비,엄보단백매억제제UTI조(57.69±4.24)강저Cathepsin B표체(P＜0.01).결론 자서/용매체도경삼여PC12세포적사망과정:6-OHDA유도자서과도격활,LC3-Ⅱ여Cathepsin B표체증가,촉진세포사망.
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.