- 學位論文
靛花青素光動力治療對大鼠頸動脈狹窄及平滑肌細胞之抑制作用探討
Inhibitory Effect of Indocyanine Green Mediated Photodynamic Therapy on Restenosis of Rat Carotid Arteries and Smooth Muscle Cells
摘要
光動力治療 (Photodynamic therapy, PDT) 近年來已被廣泛探討於治療腫瘤與非癌症疾病中，PDT 的基礎在於光感劑，累積在病變處，再以適合波長之光能活化光感藥物，使其產生具細胞毒性的活性氧物質，以達到標靶性毒殺細胞的功效。本研究中首先證實波長 780 nm 之發光二極體光照，搭配靛花青素 (Indocyanine Green, ICG) 進行PDT，著實能抑制頸動脈內皮增生。接著以體外的方式，探討大鼠動脈平滑肌 A-10 細胞接受 PDT 後的效應與相關機制。將 A-10 細胞與不同濃度的 ICG 共培養，在倒立式顯微鏡下可以觀察到 ICG濃度增加，細胞內藥物的綠色累積隨之增加；濃度 20 μM 共培養 8 小時後有最高的累積。不同濃度的 ICG 與 A-10 細胞共培養 8 小時之後進行光照 (4 J/cm2)，細胞存活率分析結果，得到半抑制劑量為 7.73 μM。以乳酸脫氫酶 (Lactate dehydrogenase, LDH) 釋放分析，結果發現 A-10 細胞與 9 μM 共培養，未照光之 LDH，釋放率小於 5%，顯示不具有暗毒性，PDT 之後釋放率大於 50%，與細胞存活率分析結果相互呼應。丙二醛試驗結果發現脂質過氧化程度隨之增加。Hochest 33258 螢光染色發現在光照 2 J/cm2 下有染色質濃縮現象，且隨光照劑量增加更明顯。PDT 後 45 分鐘進行 Annexin V/Propidium iodide 螢光染色，發現 A-10 細胞在 PDT 後大多發生細胞凋亡；進一步以流式細胞儀檢測，結果顯示，大部分細胞走向晚期凋亡。PDT 後 12 小時，可以觀察到細胞週期中亞二倍體 (Sub-G1) 比例的提升。西方點墨法探討 PARP 和 Caspase-3 的表現量，結果發現，分子量為 25 kDa 的 PARP 在 PDT 後有明顯的條帶；Caspase-3 在 PDT 組表現量下降。本研究結果顯示，ICG 介導 PDT 可使 A-10 細胞發生細胞凋亡，進而抑制大鼠頸動脈狹窄現象。
並列摘要
Photodynamic therapy (PDT) has been widely investigated in recent years in the treatment of cancer and non-cancer diseases. The basis of PDT is the accumulation of photosensitizers in the lesion site and activated by light of suitable wavelength which produced cytotoxic reactive oxygen species to achieve target toxic cytotoxicity. In this study, we first demonstrated that PDT combined with Indocyanine Green (ICG) using 780 nm light emitting diode irradiations could effectively inhibit carotid endothelial hyperplasia. Next, we studied the PDT effect on rat aortic smooth muscle cells (A-10). A-10 cells were incudated with different concentrations of ICG. The inverted microscope observation showed accumulation of green color increased when increasing the concentration of ICG. Highest accumulation was found of 8 h incubated at a concentration of 20 μM. Different concentrations of ICG were incubated with A-10 cells for 8 hours and then submitted to light irradiation (4 J / cm2). The results of cell viability assay showed that the half-inhibitory dose was 7.73 μM. Lactate dehydrogenase (LDH) release analysis revealed that A-10 cells incubated with 9 μM ICG without light irradiation resulted in less than 5% LDH release, which indicted no dark toxicity. While PDT resulted in more than 50% LDH release. LDH release result corresponded to cell viability result. Malondialdehyde test after PDT showed the degree of lipid peroxidation increased. Hochest 33258 fluorescence staining revealed PDT caused chromatin condensation 2 J/cm2 of light irradiation and was more pronounced with increasing light exposure. Forty-five minutes after PDT, Annexin V/Propidium iodide fluorescent staining found that both neciosis and apoptosis of A-10 cells occurred after PDT. Flow cytometry showed that most of the cells were late apoptotic cells. At 12 hours after PDT, a rise in the sub-diploid (Sub-G1) ratio in the cell cycle was observed. Western blotting studies of PARP and Caspase-3 performance revealed that PARP with a molecular weight of 25 kDa had a clear band after PDT; Caspase-3 expression decreased in the PDT group.The results of this study show that ICG mediated PDT can induce apoptosis of A-10 cells and further inhibit carotid artery stenosis in rats.