焦脱镁叶绿酸-a甲酯介导的光动力对人骨肉瘤MG63细胞凋亡影响的研究

发布时间：2018-07-02 19:35

本文选题：MPPa + 光动力疗法　；参考：《重庆医科大学》2017年硕士论文

【摘要】：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(0.75μmol/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(P0.05);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)(P0.05);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(P0.05)。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(P0.05)。结论:MPPa-PDT可诱导人骨肉瘤MG63细胞发生凋亡,且内质网应激反应参与了MPPa-PDT诱导细胞凋亡的过程。
[Abstract]:Objective: to investigate the effect of photodynamic therapeutic therapy (photodynamic therapeutic therapy) on apoptosis of human osteosarcoma MG63 cells in logarithmic phase. Methods: human osteosarcoma MG63 cells in logarithmic phase were divided into 4 groups. They were blank control group (control group), MPPA drug group (MPPA group), light group (LED group), MPPa-PDT experimental group (MPPa-PDT group). MPPa-PDT group and MPPA group were added MPPA (0.75 渭 mol / L) in dark condition. The cells of LED group and MPPa-PDT group were exposed to integrated LED special light source for 120 s (dose 4.8 J / cm2). After illumination, cell morphology was observed by inverted phase contrast microscope, and cell apoptosis was observed by Hoechst33258 staining under transmission electron microscope. Apoptosis rate and intracellular calcium level were detected by flow cytometry. P-PERK, C / EBP homologous protein chop and cleaved-Caspase-12 were detected by Western blot. Results under the inverted phase contrast microscope, Hoechst33258 staining showed typical apoptotic morphological changes, such as nuclear pyknosis and fragmentation. The apoptotic rate detected by flow cytometry was 48.76% 卤3.54, which was significantly higher than that in the control group (5.04% 卤0.41g) MPPA group (5.33% 卤0.38%) and LED group (6.48% 卤0.46) (P0.05), and the intracellular calcium ion level was 485.29 卤58.77, which was significantly higher than that in the control group (97.24 卤4.77) MPPA group (97.95 卤6.30) LED group (101.17 卤5.26) (P0.05). The relative expression of p-PERKG CHOPOPcleaved-Caspase-12 was significantly higher than that of the other three groups after 6 h culture (P0.05). There were no significant changes in apoptotic morphology and endoplasmic reticulum (ER) morphology in MPPA group and LED group in control group. There was no significant difference between the two groups (P0.05). Conclusion the apoptosis of human osteosarcoma MG63 cells can be induced by 1: MPPa-PDT, and endoplasmic reticulum stress is involved in the process of MPPa-PDT induced apoptosis of human osteosarcoma MG63 cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R738

【参考文献】