臺灣博碩士論文加值系統

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。

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.

摘要I
AbstractII
致謝IV
目錄V
圖目錄VIII
表目錄X
縮寫表 XI
第一章 緒論 1
1.1.前言 1
1.2.理論基礎 3
1.2.1.光動力治療原理 3
1.2.2.平滑肌細胞與血管增生之關係 5
第二章 文獻回顧 7
2.1.PDT 應用於血管病變 7
2.2.靛花青素應用於光動力治療 8
2.3.靛花青素運用於血管病變 10
2.3.研究動機與目的 10
第三章 材料與方法 14
3.1.體外實驗 14
3.1.1.細胞來源與特性 14
3.1.2.細胞培養藥物之配製 15
3.1.3.細胞繼代 16
3.1.4.細胞凍存 16
3.1.5.細胞解凍 17
3.1.6.光動力治療 17
3.1.7.細胞與藥物共培養之型態觀察 20
3.1.8.細胞對藥物攝取量分析 20
3.1.9.蛋白質定量 21
3.1.10.細胞存活率分析與半抑制劑量的探討 22
3.1.11. 乳酸脫氫酶釋放率分析 23
3.1.12.丙二醛產生量分析 24
3.1.13.Hoechst 33258 螢光染色分析 26
3.1.14.Alexa Fluor® 488 Annexin V/ Propidium Iodide 螢光染色分析 27
3.1.15.Alexa Fluor® 488 Annexin V/ Propidium Iodide 染色流式細胞儀分析 28
3.1.16.細胞週期分析 29
3.1.17.西方點墨法 30
3.2.體內實驗 43
3.2.1.實驗動物來源與飼養 43
3.2.2.大鼠血管內壁增生模型 43
3.2.3.光動力治療 45
3.2.4.光動力治療之動物實驗方法 47
3.2.5.組織學分析 48
3.2.6.組織處理 48
3.2.7.組織脫水 49
3.2.8.組織包埋 50
3.2.9.組織切片 50
3.2.10.組織染色 50
3.2.11.影像分析 51
3.3.統計分析 51
第四章 結果 52
4.1. 細胞與 ICG 共培養之型態分析 52
4.2. A-10 細胞對靛花青素的藥物攝取量試驗 54
4.3.細胞存活率分析與半抑制劑量的探討 56
4.4.LDH 釋放率 58
4.5. MDA 產生量分析 60
4.6.螢光染色分析 61
4.7.Alexa Fluor® 488 Annexin V/ Propidium Iodide 染色流式細胞儀分析 65
4.8.細胞週期分析 67
4.9.凋亡蛋白表現 69
4.10.光動力治療頸動脈狹窄大鼠 71
4.10.1.頸動脈血管厚度分析 75
4.10.2.頸動脈血管管徑分析 77
4.10.3.頸動脈血管壁面積分析 79
4.10.4.頸動脈血管壁增生面積 81
第五章 討論 83
5.1. ICG 在 A-10 細胞內的累積與藥物攝取 83
5.2. ICG 對 A-10 細胞進行 PDT 的存活率與死亡率的影響 83
5.3. ICG 對 A-10 細胞進行 PDT 的 MDA 含量影響 85
5.4. ICG 對 A-10 細胞進行 PDT 的死亡特性 85
5.5. ICG 對 A-10 細胞進行 PDT 的凋亡蛋白表現 85
5.6. PDT 對大鼠左外頸動脈增生模型之評估 86
第六章 結論 88
附錄 89
I.儀器表 89
II.藥品表 91
參考文獻 94

圖目錄
圖 1- 1 PDT 機制圖 4
圖 2- 1 研究架構圖 13
圖 3- 1 A-10 細胞型態 14
圖 3- 2 體外光源設備 18
圖 3- 3 蛋白質定量曲線 22
圖 3- 4 大鼠頸動脈氣球擴張術流程 45
圖 3- 5 體內光源設備 46
圖 3- 6 豬皮試驗 47
圖 3- 7 大鼠進行 PDT 流程 48
圖 4- 1 A-10 細胞與不同濃度的 ICG 共培養 24 小時之細胞型態 53
圖 4- 2 A-10 細胞與 ICG 共培養不同時間之攝入量 55
圖 4- 3 24 小時，A-10細胞以 ICG 進行 PDT 之細胞存活率 57
圖 4- 4 A-10 細胞以 ICG 進行 PDT 之 LDH 釋放量 59
圖 4- 5 ICG PDT 對A-10 細胞的 MDA 含量影響 61
圖 4- 6 ICG PDT 對 A-10 細胞凋亡反應表現 63
圖 4- 7 ICG PDT 對 A-10 細胞的染色質濃縮影響 64
圖 4- 8 ICG PDT 對 A-10 細胞的染色質濃縮影響 66
圖 4- 9 ICG PDT 對 A-10 細胞的週期影響 68
圖 4- 10 凋亡蛋白的表現 70
圖 4- 11 正常大鼠與氣球擴張術處理大鼠的頸動脈組織切片 72
圖 4- 12 頸動脈狹窄大鼠接受 PDT 後之頸動脈組織切片 73
圖 4- 13 頸動脈狹窄大鼠接受 PDT 後之頸動脈厚度分析 76
圖 4- 14 頸動脈狹窄大鼠接受 PDT 後之頸動脈管徑分析 78
圖 4- 15 頸動脈狹窄大鼠接受 PDT 後之頸動脈血管壁面積分析 80
圖 4- 16 頸動脈狹窄大鼠接受 PDT 後之頸動脈血管壁增生面積分析 82
圖 5- 1 ICG-PDT 對 A-10 細胞之存活率與 LDH 釋放率的關係 84
圖 5- 2 ICG 進行 PDT 誘導 A-10 細胞的可能死亡路徑 87

表目錄
表3- 1 A-10 細胞基本資料 15
表3- 2 本研究實驗參數表 19
表3- 3 蛋白質標準曲線濃度稀釋表 21
表3- 4 MDA Assay 藥物配製表 25
表3- 5 1N H2SO4 水解 TMP標準曲線濃度稀釋表 26
表3- 6 5X Annexin-binding buffer 配製表 27
表3- 7 Homo Buffer 配製表 32
表3- 8 4X Sample Buffer 配製表 33
表3- 9 膠體溶液配製表 34
表3- 10 SDS-PAGE膠體配製表 35
表3- 111X Running buffer配製表 36
表3- 12 Towbin transfer buffer配製表 37
表3- 13 Ponceau S配製表 37
表3- 14 Coomassie Blue R-250 染劑配製表 38
表3- 15 5 % 阻斷液配製表 39
表3- 16 抗體稀釋比例 39
表3- 17 Tris-buffered saline with Tween-20 (TBST) 溶液配置表 39
表3- 18 Stripping Buffer 溶液配置表 41
表3- 19 體內實驗參數表 42
表4- 1半抑制劑量 56

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