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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) has gradually become a new alternative treatment of ovarian cancer in recent years. Hematoporphyrin monomethylether (HMME) is a new type of photosensitizer developed independently in our country. The purpose of this experiment is to study the fluorescence microscopy of HMME in ovarian cancer cell line SKOV3 in vitro and to verify its effect on SKOV3 Photodynamic killing effect. Methods: After incubated with 30μg / ml HMME for different time, the fluorescence and intracellular localization of HMME were detected by fluorescence microscopy and confocal laser scanning microscopy. The fluorescence intensity of HMME was analyzed by morphological software. The fluorescence intensity of HMME at different concentrations (5 ~ 50μg / ml ) HMME was incubated with the cells for 3 hours and then irradiated with different energy laser (1.5, 3, 6, 12 J / cm2). Cell viability was detected by MTT colorimetric assay. cm2, respectively. The cells were harvested at 4 and 24 hours and AnnexinV / PI double staining was performed. The cell death patterns were analyzed by flow cytometry. Results: HMME diffusely distributed in the cytoplasm with red fluorescence, and the intracellular fluorescence intensity peaked at 3h. High concentrations of HMME alone may have dark toxicity on cells, whereas laser irradiation alone has no effect on cell survival. With the increase of HMME concentration and laser dose, the cell survival rate decreased gradually. When the HMME concentration increased to ≥ 40μg / ml, increasing the drug concentration and laser dose of cell survival rate does not decrease. Flow cytometry analysis showed that HMME photodynamic treatment of dead cells are mainly necrotic cells. Conclusion: HMME has a photodynamic killing effect on SKOV3 cells.