5-ALA-PDT联合放疗对常氧与乏氧人卵巢癌SKOV3细胞的协同杀伤作用机制

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48 h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）。0 h、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。

Synergetic killing mechanisms of 5-ALA-PDT in combination with radiotherapy on human ovarian carcinoma SKOV3 cells under normoxia and hypoxia

OBJECTIVE To study synergetic killing mechanisms of 5-aminolevulinic acid (5-ALA) mediated photodynamic therapy (PDT) (5-ALA-PDT) in combination with radiotherapy on human ovarian carcinoma SKOV3 cells under normoxia and hypoxia.METHODS Normoxia group and hypoxia group were divided,and each had normal control (N),drug treatment alone (0.3 mmol·L^-1),radiation treatment alone (0.3 mmol·L^-1+6 Gy),PDT (0.3 mmol·L^-1+0.5 J·cm^-2) and combined therapy (6 Gy+0.3 mmol·L^-1-0.5 J·cm^-2).Fluorescence microscopy was applied to observe fluorescence intensity of protoporphyrin IX converted by 5-ALA,trypan blue staining and inverted microscope to detect cell membrane damage,Hoechst 33342 staining method with fluorescence microscopy to detect apoptosis,ROS kits combined flow cytometry to detect ROS levels.RESULTS After 4 h,red fluorescence obviously existed nearby cell membrane.Fluorescence intensity under normoxia was markedly stronger than under hypoxia.After 24 h or 48 h,membrane breakage rates of combined therapy under normoxia were higher than under hypoxia (P<0.01),and both of them were increasing with time (P<0.01).Membrane breakage rates of combined therapy were higher than those of radiation treatment alone and PDT alone at the same time and within the same group (P<0.01).After 24 h or 48 h,apoptosis rates of combined therapy under normoxia was less than under hypoxia at 24 h (P<0.01),but not significant at 48 h (P>0.05),and both of them were decreasing (P<0.01) with time.Apoptosis rates of combined therapy were higher than those of radiation dosing group and PDT group alone at the same time and within the same group at 24 h,but were opposite at 48 h (P<0.05).After 0 h,12 h or 24 h,ROS levels of combined therapy under normoxia were lower than under hypoxia (P<0.01),and both of them increased firstly and then decreased (P<0.01) over time,and were the highest at 12 h.ROS levels of combined group were higher than those of radiation dosing group and PDT group alone at the same time and within the same group (P<0.05).CONCLUSION Under normoxia,synergistic effects showed by 5-ALA-PDT in combination with radiation may be related to ROS production induced membrane rupture which caused abundant cell necrosis,while synergistic effects under hypoxia may be related closely with abundant cell apoptosis induced by ROS.