- 學位論文
光動力治療對小鼠乳腺癌 EMT6 細胞之細胞遷移及 EMT6 腫瘤小鼠治療之研究
The Studies of Photodynamic Therapy in Migration of Murine Mammary Carcinoma EMT6 Cells and EMT6 Tumor-bearing Mice
摘要
光動力療法 (Photodynamic therapy, PDT) 為使用光感藥物累積於特定的組織中，給予特定波長的光源，激發光感藥物產生光化學反應，產生活性氧分子，對癌症組織產生傷害，以達到癌症治療的目的。本研究探討脫鎂葉綠酸 a (Pho a) 搭配波長 660 nm 發光二極體光照對小鼠乳腺癌 EMT6 細胞及乳癌小鼠腫瘤之影響。首先，利用二甲基硫醇二苯基四唑溴 (MTT) 分析得知 Pho a在不同共培養時間以及不同光照能量下對 EMT6 細胞的半致死劑量 (LD50)。以 2.55 J/cm2 能量進行 PDT 的藥物 LD50 為 8.2 ± 3.5 ng/mL；光照能量為 5.10 J/cm2 的 LD50 降為 3.5 ± 0.2 ng/mL。傷口癒合試驗評估 Pho a-PDT 後對細胞移動能力之影響，結果發現，藥物共培養 2 小時後進行 PDT 不影響細胞移動能力；共培養時間增加至 4 小時，光照劑量 5.10 J/cm2 可使細胞移動能力增加；當共培養時間增加至 6 小時，2.55 J/cm2 及5.10 J/cm2 照射皆可促進細胞移動。進一步測試細胞跨膜能力及侵入試驗，結果顯示 Pho a 共培養時間以及光照能量增加可抑制細胞跨膜能力及侵襲能力。在 PDT 後，EMT6 細胞出現細胞核腫脹、Annexin V/碘化丙啶雙重染色等細胞壞死現象，且共培養時間越長、光照能量越高，則壞死現象越明顯。利用尾靜脈注射 Pho a 及光照 10.2 J/cm2 對 EMT6 腫瘤小鼠進行 PDT，可減緩腫瘤生長速度，而分次光照 (Fractional -Photodynamic Therapy, F- PDT) 組治療效果更佳。綜上所述，Pho a-PDT 對 EMT6 細胞可有效造成細胞的光敏毒性，對 EMT6 腫瘤小鼠則可以減緩腫瘤之生長。本研究顯示 Pho a-PDT可作為癌症的輔助治療。
並列摘要
Photodynamic therapy (PDT) is a modality for cancers using photosensitizers that are specifically accumulated in diseased tissue. Upon exposure to light with specific wavelength, they produce reactive oxygen species and become toxic to targeted malignant cells. The aim of this study was to investigate the PDT effects on EMT6 murine mammary cancer cells and EMT6-tumor bearing mice in the presence of pheophorbide a (pho a) and 660 nm light emitting diodes (LED) irradiation. First, half lethal dose (LD50) under different incubation time with pho a and different light dose was obtained by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. LD50 is 8.2±3.5 ng/mL with radiant exposure of 2.55 J/cm2 and 3.5±0.2 ng/mL with radiant exposure of 5.10 J/cm2. Wound healing assay was used to determine the effect Pho a-PDT on cell migration. The results showed that PDT after 2-hr incubation with Pho a had no effect on EMT6 cell migration. Incubation time of 4 hours and radiant exposure of 5.10 J/cm2 increased cell migration ability. Six-hour coculture and radiant exposure of 2.55 J/cm2 also increased cell migration ability. Next, transwell cell migration and invasion assays showed that transmembrane migration and invasion ability decreased with the increase of coculture time with Pho a and light dose. EMT6 cells exhibited the phenomenon of necrosis such as swollen nuclei and double staining with annexin/propidium iodide after PDT. Necrotic cell population increased with the increase of coculture time with Pho a and light dose. EMT6-bearing mice injected 2 mg/mL Pho a by tail vein, and irradiated with 10.2 J/cm2 exhibited slower tumor growth. Fractional-Photodynamic therapy (F-PDT) further decreased the tumor growth. In summary, Pho a-PDT possesses cytotoxicity on EMT6 cells, and slows down tumor growth in EMT6-bearing mice, which may be used as an adjunct therapy for cancer treatment.