自噬和凋亡联合在ALA-PDT诱导C6胶质瘤细胞死亡中的作用机制

Combination mechanisms of autophagy and apoptotic in ALA-PDT induced death of C6 glioma cells

目的探讨自噬和凋亡在氨基酮戊酸光动力疗法(ALA-PDT)诱导大鼠C6胶质瘤细胞死亡中的联合作用机制。方法通过MDC染色和电镜等观察ALA-PDT后的自噬现象,检测Caspase-3活化以探讨自噬与凋亡机制在ALA-PDT诱导C6胶质瘤细胞死亡中的作用机制。结果 ALA-PDT后3 h,MDC染色C6胶质瘤细胞内可见明显的点状荧光颗粒,且随时间逐渐明显增多,24 h时荧光颗粒变弱。透射电镜观察到ALA-PDT后3 h时,胞浆中即出现大量独立双层膜结构,12 h时自噬体及其独立双层膜结构相对变少,24 h时观察到典型的凋亡形态,同时仍可见较多的自噬体和独立双层膜结构及自噬溶酶体的出现。Western-blot结果提示,ALA-PDT后1 h C6胶质瘤细胞内少量Caspase-3活化,12 h时Caspase-3酶原表达最高;AcDEVD-CHO干预后Caspase-3酶原活化明显减弱。结论凋亡机制有助于ALA-PDT诱导C6胶质瘤细胞的死亡,而自噬的激活延迟ALA-PDT诱导的细胞凋亡。

Objective To explore the combination mechanisms of the autophagy and apoptosis in aminolevulinic acid photodynamic therapy （ALA-PDT） induced death of rat with C6 glioma cell death.Methods The autophagy was observed after ALA-PDT by MDC staining and electron microscopy,and Caspase-3 activation was detected in order to investigate the mechanism of autophagy and apoptosis in ALA-PDT induced death of C6 glioma cell.Results 3 hours after ALA-PDT,the MDC staining C6 glioma cells showed significant punctate fluorescent particles and gradually increased significantly.The fluorescent particles became weak at 24 hours.3 hours after ALA-PDT,the cytoplasm appeared largely independent bilayer structure,autophagy and its independent double membrane structure became relatively less at the time of 12 hours,and 24 hours when typical apoptotic form was observed,while still more visible and independent autophagy bilayer structure and lysosomal autophagy occurred.Western-blot results suggested that 1 hour after ALA-PDT,C6 glioma cells within a small activation of Caspase-3,Caspase-3 zymogen highly expressed when 12 hours.Ac-DEVD-CHO Caspase-3 after the intervention significantly reduced plasminogen activator.Conclusion Apoptotic mechanisms contribute to ALA-PDT induced death of C6 glioma cells,and autophagy activation delays ALA-PDT induced apoptotic cells.