- 學位論文
脫鎂葉綠素 a 在 4T1 腫瘤小鼠之光動力治療及藥物動力學研究
The Studies of Photodynamic Activities and Pharmacokinetics of Pheophytin a in 4T1 Tumor-bearing Mice
摘要
光動力療法 (Photodynamic therapy, PDT) 係使光感藥物選擇性的累積於特定組織或腫瘤細胞，施與特定波長之光能激發光感藥物使之發生光化學反應，產生具有細胞毒性的活性氧分子，對腫瘤局部造成損傷，以達到癌症治療的目的。本研究目的在探討脫鎂葉綠素搭配 660 nm 發光二極體照射對小鼠乳腺癌 4T1 細胞及 4T1 腫瘤Balb/c小鼠之 PDT 療效。首先觀察到脫鎂葉綠素 a 與 4T1 細胞共培養 2 小時，細胞內累積濃度可達最高點；接著發現隨著藥物濃度提升、共培養時間增加及光照能量增強，PDT 對 4T1 細胞的致死率也隨之增加。在共培養 2 小時，光照 2.55 J/cm2，脫鎂葉綠素 a 的半致死劑量 (LD50) 為 459.3 ± 28.9 ng/mL；光照 5.10 J/cm2，LD50 降為 320.2 ± 23.3 ng/mL。同樣的參數下，脫鎂葉綠素 b 的 LD50 分別為 303.8 ± 25.1 及 226.7 ± 5.4 ng/mL。TUNEL 分析可發現隨著共培養時間由 2 小時增長到 6 小時，凋亡細胞比例亦隨之增加。對小鼠進行劑量為 87.25 mg/kg 的急毒性測試，結果發現脫鎂葉綠素 a 注射 14 天內不會造成小鼠體重下降，主要器官的組織切片也無明顯損傷；同時，連續14天注射 2 mg/kg 脫鎂葉綠素 a 對腫瘤小鼠也無亞急毒性。進一步觀察尾靜脈注射脫鎂葉綠素 a在正常小鼠與 4T1 腫瘤小鼠體內的生物分佈狀態，發現注射 4 小時後，在正常鼠體內，以肝臟所累積的濃度最高；而在 4T1 腫瘤小鼠，則以腫瘤所累積的濃度最高。每天注射 2 mg/kg 脫鎂葉綠素 a，以10.2 J/cm2 光照進行 PDT 可顯著抑制4T1 腫瘤小鼠腫瘤組織的生長；組織染色分析驗證 PDT 組出現較多的凋亡細胞及腫瘤組織損傷。綜上所述，脫鎂葉綠素 a 能選擇性的累積在腫瘤組織，並對小鼠乳腺癌 4T1 細胞具有光敏毒性，結合光照能抑制乳癌小鼠腫瘤組織的生長。
並列摘要
Photodynamic therapy (PDT) is an alternative modality for cancer treatment. Tumor site is damaged by cytototoxic reactive oxygen species (ROS) produced by the activated photosensitizer selectively accumulated in cancerous cells and specific tissues after light irradiation with specific excitation wavelength. The aim of this study was to the efficacies of pheophytin mediated PDT combined with 660 nm light emitting diodes (LEDs) against murine mammary gland adenocarcinoma 4T1 cells and 4T1 tumor bearing Balb/c mice. First of all, the maximal concentration of pheophytin a in 4T1 cells was found 2 hrs after incubation with the photosensitizer. Cell viability decreased as the photosensitizer concentration, incubation time and light dose increased. When incubated with pheophytin a for 2 hrs, half lethal dose (LD50) for 4T1 cells was 459.3 ± 28.9 ng/mL with 2.55 J/cm2 irradiation and 320.2 ± 23.3 ng/mL with 5.10 J/cm2 irradiation. For pheophytin b, LD50 was 303.8 ± 25.1 and 226.7 ± 5.4 ng/mL with 2.55 and 5.10 J/cm2 irradiation, respectively. TUNEL assay revealed that the percentage of apoptotic cells increased as the increase of incubation time with pheophytin a. Acute toxicity was studied in mice with single dose injection of 87.25 mg/kg pheophytin a. It was found that bodyweight of mice did not decline and no major organs damage was observed in tissue section within 14 days post photosensitizer administration. No subacute toxicity was seen in 4T1 tumor bearing mice after daily injection of 2 mg/kg pheophytin a. Biodistribution of pheophytin a in normal and 4T1 tumor bearing mice were analyzed after intravenous injection through the tail vein. At 4 hr post-injection, pheophytin a accumulated most in the liver of the normal mice, whereas it accumulated most at tumor site of tumor bearing mice. Tumor growth was inhibited in 4T1 tumor-bearing mice with daily intravenous injection of pheophytin a at the concentration of 2 mg/kg and light dose of 10.2 J/cm2. Histochemical staining further showed that more apoptotic cells and more tumor tissue damage post-PDT. To sum up, this study demonstrated that pheophytin a is selectively accumulated in tumor tissue, possesses phototoxicity against urine mammary gland adenocarcinoma 4T1 cells, and effectively inhibits tumor growth when combined with red light irradiation.