Objective: To explore the effect of pyropheophorbide-a methyl ester-mediated photodynamic therapy (MPPa-PDT) on the apoptosis in human osteosarcoma cell line MG63 and the underlying mechanism.
Methods: Human osteosarcoma MG63 cells in logarithmic growth phase were divided into 4 groups: blank control group (control group), the MPPa treatment group (MPPa group), the light irradiation group (LED group), and MPPa-PDT treatment group (MPPa-PDT group). MPPa-PDT group and MPPa group were incubated with MPPa (0.75?μmol/ L) for 20 hours in dark condition; control group and LED group were incubated with equal volume of fresh medium for 20 hours in the same condition. After washing with PBS and replacement with fresh culture medium, LED group and MPPa-PDT group cells were exposed to light (4.8 J/cm2) for 120 seconds. After light exposure, all groups were cultured in dark condition again. Then cellular morphology changes were observed by an inverted phase contrast microscopy, endoplasmic reticulum morphology changes were observed by transmission electron microscopy, cellular apoptosis was detected by Hoechst33258 nuclear staining, cell apoptotic rate and the levels of Ca in cells were analyzed by flow cytometry, the expression of p-PERK, C/EBP homologous protein (CHOP), cleaved-Caspase-12 were assayed by Western blot.
Results: In MPPa-PDT group, the retracted and round cells were observed; Hoechst33258 nuclear staining showed nuclear condensation, fragmentation, and other typical apoptotic morphological changes; the cell apoptotic rate (48.76%±3.54%) was significantly higher than that of control group (5.04%±0.41%), MPPa group (5.33%±0.38%), and LED group (6.48%±0.46%) (P < 0.05); the levels of Ca2+ in cells (485.29±58.77) was also significantly higher than that of control group (97.24±4.77), MPPa group (97.95±6.30), and LED group (101.17±5.26) (P < 0.05); swelling endoplasmic reticulum was observed under transmission electron microscope; the expressions of p-PERK, CHOP, and cleaved-Caspase-12 gradually increased at 1, 3, and 6 hours after treatment respectively, which were significantly higher than those of the other groups (P < 0.05). There was no typical apoptotic morphological changes and endoplasmic reticulum morphological changes in control group, MPPa group, and LED group, and there was no significant difference in the above indexes among 3 groups (P > 0.05).
Conclusions: MPPa-PDT can significantly induce apoptosis in MG63 cells. The endoplasmic reticulum stress pathway is involved in the MPPa-PDT induced apoptosis.
目的: 探讨焦脱镁叶绿酸-a甲酯（pyropheophorbide-a methyl ester，MPPa）介导的光动力疗法（photodynamic therapy，PDT）对人骨肉瘤MG63细胞凋亡的影响及其作用机制。.
方法: 取对数生长期人骨肉瘤MG63细胞分为4组，分别为空白对照组（对照组）、单纯MPPa药物组（MPPa组）、单纯光照组（LED组）以及MPPa-PDT实验组（MPPa-PDT组）。MPPa-PDT组和MPPa组于避光条件下加入MPPa（0.75 μmol/L），对照组及LED组加入等量完全培养基；避光孵育20 h后，LED组及MPPa-PDT组细胞接受集成LED特种光源照射120?s （剂量为4.8 J/cm2）。光照结束后，于避光条件下继续培养，倒置相差显微镜观察细胞形态变化，透射电镜观察内质网形态变化，Hoechst33258 染色观察细胞凋亡，流式细胞术检测细胞凋亡率及细胞内钙离子水平，Western blot 检测p-PERK、内质网源性转录因子（C/EBP homologous protein，CHOP）、活化半胱氨酸蛋白酶12（cleaved-Caspase-12）表达水平。.
结果: MPPa-PDT组倒置相差显微镜下见细胞明显回缩变圆；Hoechst33258染色示细胞核固缩、碎裂等典型凋亡形态学改变；流式细胞术检测细胞凋亡率为48.76%±3.54%，明显高于对照组5.04%±0.41%、MPPa组5.33%±0.38%及LED组6.48%±0.46% （P < 0.05）；细胞内钙离子水平为485.29±58.77，显著高于对照组（97.24±4.77）、MPPa组（97.95±6.30）、LED组(101.17±5.26)（P < 0.05）；透射电镜下见细胞内质网明显肿胀；Western blot检测示p-PERK、CHOP、cleaved-Caspase-12相对表达量分别于培养1、3、6 h后明显高于其他3 组（P < 0.05）。对照组、MPPa组及LED组细胞无显著凋亡形态改变和内质网形态改变，上述检测指标组间比较，差异均无统计学意义（P > 0.05）。.
结论: MPPa-PDT可诱导人骨肉瘤MG63细胞发生凋亡，且内质网应激通路参与了MPPa-PDT诱导细胞凋亡的过程。.
Keywords: Apoptosis; Osteosarcoma; Photodynamic therapy; Pyropheophorbide-a methyl ester.