血卟啉单甲醚对卵巢癌细胞系SKOV3的光动力学杀伤效应

Photodynamic Effect of Hematoporphyrin Monomethyl Ether on Ovarian Cancer Cell Line SKOV3

背景与目的:光动力学治疗(photodynamictherapy,PDT)近年来逐渐成为一种新的可供选择的卵巢癌治疗手段。血卟啉单甲醚(hematoporphyrinmonomethylether,HMME)是我国自主开发研制的一种新型光敏剂,本实验目的在于体外实验研究HMME在卵巢癌细胞系SKOV3细胞中的荧光显微成像,并验证其对SKOV3的光动力杀伤作用。方法:30μg/mlHMME与细胞孵育不同时间后,置于高灵敏度荧光显微镜与激光共聚焦显微镜下检测HMME荧光及其细胞内的定位,形态学软件分析其荧光强度;不同浓度(5～50μg/ml)HMME与细胞孵育3h后,以不同能量激光(1.5、3、6、12J/cm2)照射,MTT比色法检测细胞的存活率;以5μg/ml、3J/cm2,20μg/ml、6J/cm2剂量处理细胞,4h及24h后分别收集细胞行AnnexinV/PI双染色,流式细胞仪分析细胞死亡方式。结果:HMME呈红色荧光弥漫性分布于细胞浆内,细胞内荧光强度于用药3h后达到高峰。高浓度HMME单独使用可能对细胞具有暗毒性,而单独激光照射对细胞存活无影响。随着HMME浓度与激光剂量的增加,细胞存活率逐渐下降。当HMME浓度增高到≥40μg/ml时,加大药物浓度与激光剂量细胞存活率并不随之降低。流式细胞仪分析结果显示HMME光动力学处理后死亡细胞主要为坏死细胞。结论:HMME对SKOV3细胞具有光动力学杀伤效应。

BACKGROUND ＆ OBJECTIVE; Photodynamic therapy （PDT） is a new treatment choice for ovarian carcinoma. Hematoporphyrin monomethyl ether （HMME） is a novel photosensitive reagent developed in China. This study was to investgate the photodynamic effect of HMME- based PDT on human epithelial ovarian cancer cell line SKOV3. METHODS. After an incubation with 30 μg/ml HMME for different time, the fluorescent image and intracellular location of HMME in SKOV3 cells were observed under a fluorescent microscope and laser scanning confocal microscope （LSCM）. After being treated with different doses （5-50 μg/ml） of HMME and irradiated with different optical doses （1.5-12 J/cm^2） of laser, the survival rate of SKOV3 cells was measured by MTT assay. Mechanisms of cell death during PDT was determined by Annexin V/PI double staining technique and analyzed by flow cytometry. RESULTS; Red fluorescence appeared shortly after administration of HMME and localized in cytoplasm; intracellular fluorescence intensity reached the peak after 3 h. High concentrations of HMME alone had cytotoxicity to SKOV3 cells, while laser irradiation alone had no effect on cell survival. Survival rate of SKOV3 cells was gradually decreased along with the increase of HMME concentration and laser dose, but such a trend diminished when HMME concentration reached 40 μg/ml. After treatment of HMME, the dead cells were predominantly necrosis cells. CONCLUSION, HMME has a photodynamic effect on SKOV3 cells.