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目的:探讨在常氧与乏氧下5-氨基酮戊酸(5-ALA)介导的光动力疗法(PDT)(5-ALA-PDT)联合放疗对人卵巢癌SKOV3细胞的协同杀伤作用机制。方法:实验分为常氧组与乏氧组,两组再各设正常对照组(N)、单纯药物组(0.3 mmol·L-1)、放射加药组(0.3 mmol·L-1+6 Gy)、光动力组(0.3 mmol·L-1+0.5 J·cm-2)及联合组(6 Gy+0.3 mmol·L-1-0.5 J·cm-2),采用荧光显微镜观察5-ALA转化为原卟啉IX的荧光强度,倒置显微镜结合台盼蓝单染法检测细胞膜破损率,荧光显微镜结合Hoechst 33342单染法检测细胞凋亡,流式细胞仪结合试剂盒检测ROS含量。结果:4 h后细胞膜附近明显出现红色荧光,常氧下的荧光强度显著强于乏氧。24 h或48 h后,联合组细胞膜破损率常氧高于乏氧(P<0.01),随着时间的延长细胞膜破裂增多(P<0.01);同时间点同组内联合组细胞膜破损率比单纯放射加药组、光动力组都要高(P<0.01)。24 h或48h后,联合组细胞凋亡率24 h常氧低于乏氧(P<0.01),48 h不显著(P>0.05),随着时间的延长凋亡率不断降低(P<0.01);同时间点同组内联合组24 h细胞凋亡率比单纯放射加药组、光动力组都要高,而48 h却都要低(P<0.05)。0h、12 h或24 h后,联合组ROS含量常氧低于乏氧(P<0.01),随着时间的延长ROS含量先升高后降低(P<0.01),12 h含量最高;同时间点同组内联合组ROS含量都比单纯放射加药组、光动力组都要高(P<0.05)。结论:常氧下5-ALA-PDT与放疗联用表现出协同作用,可能与产生的ROS导致细胞膜破损造成细胞大量坏死有关;而乏氧下表现出协同作用可能与ROS导致细胞大量凋亡有密切联系。
OBJECTIVE: To investigate the synergistic cytotoxicity of 5-ALA-PDT combined with radiotherapy on human ovarian cancer SKOV3 cells under normoxia and hypoxia . Methods: The experiment consisted of normoxia group and hypoxia group. The normal control group (N), pure drug group (0.3 mmol·L-1), and radiotherapy group (0.3 mmol·L-1 + 6) Gy, 0.3 mmol·L-1 + 0.5 J · cm-2, and 6 Gy + 0.3 mmol·L-1-0.5 J · cm-2 in the photodynamic therapy group. The fluorescence intensity of protoporphyrin IX was detected by inverted microscope and trypan blue exclusion method. The apoptosis rate was detected by fluorescence microscopy combined with Hoechst 33342 single staining method. The ROS level was detected by flow cytometry combined with kit. Results: After 4 h, the red fluorescence appeared obviously near the cell membrane, and the fluorescence intensity under normoxia was significantly stronger than that under hypoxia. After 24 h or 48 h, the membrane damage rate in the combined group was higher than that in hypoxia (P <0.01), and the cell membrane rupture increased with time (P <0.01) Radiotherapy alone group were higher (P <0.01). After 24 h or 48 h, the apoptosis rate of the combined group was lower than that of the hypoxia 24 h (P <0.01), but not significantly after 48 h (P> 0.05) ); At the same time, the apoptosis rate of 24 h in the same group and in the same group was higher than those in the radiotherapy group and the photodynamic therapy group, but lower in 48 h (P <0.05). The content of ROS in the combined group was lower than that in hypoxia at 0h, 12h or 24h (P <0.01). The content of ROS increased firstly and then decreased (P <0.01) with the increase of 12h, ROS levels in the combined group were higher than those in the radiotherapy alone group and the photodynamic therapy group (P <0.05). CONCLUSION: The combination of 5-ALA-PDT and radiotherapy under normoxia shows synergistic effect, which may be related to the massive necrosis of cell caused by cell membrane rupture caused by ROS. In hypoxia, synergistic effect may be associated with ROS-induced cell apoptosis close relationship.