摘要
目的探讨芦荟大黄素(aloe emodin,AE)介导的光动力(aloe emodin-photodynamic therapy,AE-PDT)诱导骨肉瘤MG63细胞产生自噬,并探讨自噬和凋亡的关系。方法将细胞分为AE-PDT对照组(Control组、AE组、LED组)和AE-PDT实验组。AE-PDT作用于人骨肉瘤MG63细胞后,采用CCK-8法检测细胞活性,2’,7’-二氯荧光二乙酸盐(2’,7’-dichlorofluorescin diacetate,DCFH-DA)荧光检测细胞活性氧(reactive oxygen species,ROS)水平,单丹磺酰尸胺(monodansylcadaverin,MDC)染色检测细胞的自噬小体,透射电镜观察细胞的自噬体,流式细胞仪检测细胞凋亡情况,Western印迹检测自噬相关蛋白微管相关蛋白1轻链3(LC3)和Beclin-1的表达。结果 AE-PDT能明显抑制人骨肉瘤MG63细胞的活性,且其抑制作用呈芦荟大黄素浓度-光动力能量剂量依赖性。AE-PDT实验组细胞内ROS水平和自噬小体均较3个对照组增高;透射电镜下可见典型自噬体;AE-PDT能诱导细胞产生自噬和凋亡,3-甲基腺嘌呤抑制自噬后能增加细胞的凋亡率(P<0.05);Western印迹检测LC3-Ⅱ/LC3-Ⅰ和Beclin-1/β-actin的表达高于阴性对照组。结论 AE-PDT能诱导骨肉瘤MG63细胞产生自噬。AE-PDT作用早期,自噬能延缓凋亡的发生可能起保护性作用。
Objective To investigate the autophagy of osteosarcoma MG63 cells induced by aloe emodin-mediated photodynamic therapy, and to discuss the relationship between autophagy and apoptosis. Methods The cultured MG63 cells were divided into 4 groups： empty control group, AE group, LED group, and AE-PDT group. MG63 cells in AE-PDT group were treated with aloe emodin combined with photodynamic therapy. The cell viability was measured by CCK-8, the DCFH-DA probe was used to detect cell ROS level, the autophagosomes were examined by MDC staining and electron microscope, the apoptosis of MG63 cells was detected by flow cytometry, and the autophagy-related protein （LC3 and Beclin-1） expressions were assessed by Western blotting analysis. Results Photosensitizer aloe emodin-mediated PDT significantly suppressed cell viability in a photosensitizer concentration- and energy density-dependent manner. The ROS level and autophagosomes were significantly increased in the AE-PDT group, with classical autophagosomes found under the electron microscope. AE-PDT induced autophagy and apoptosis of MG63 cells. The apoptosis rate was significantly increased when autophagy was inhibited by 3-MA （P〈0.05）. The results of Western blotting analysis showed that ratios of LC3-Ⅱ/LC3-Ⅰ and Beclin-1/β-actin in AE- PDT group were higher than those in the single AE treatment group, single light irradiation group, and the empty control group. Conclusion AE-PDT can induce autophagy in the MG63 cells. At the early stage of AE-PDT treatment, autophagy can postpone apoptosis and hence exert a protective effect.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2014年第12期1347-1352,共6页
Academic Journal of Second Military Medical University
基金
国家自然科学基金(81101692)
重庆市科委自然科学基金(2011BB5136)~~
关键词
芦荟大黄素
光动力疗法
骨肉瘤
自噬
aloe emodin
photodynamic therapy
osteosarcoma
autophagy
