臺灣博碩士論文加值系統

光動力療法 (photodynamic therapy、PDT) 為非侵入式治療，將光敏劑或光敏劑前驅物以塗抹、口服或注射方式給藥，光敏劑在體內會選擇性地滯留於癌症或癌前病變組織內，以特定波長光線照射，感光劑會吸收特定波長的光能而被激發，啟動光化學反應產生自由基及單態氧造成細胞傷害，達到選擇性消滅癌細胞的作用，而其螢光特性也可用於腫瘤的診斷及定位。由於光的物理性質直接決定了治療的效果及治療的深度，因此選擇適當的感光劑、可吸收的光線波長及適合的治療時間點，是光動力療法成功的關鍵。
本研究使用已用於臨床治療之5-氨基酮戊酸 (5-aminolaevulinic acid、ALA) 光敏感劑前驅物質為治療基礎，並於倉鼠口腔癌前病變模式動物進行光動力治療之光源、功率、劑量比較，篩選出合適於倉鼠口腔癌前病變模式動物光動力治療之組合。並以局部塗抹方式比較甲氨蝶呤 (Methotrexate、MTX)、卡泊三醇 (1,24(OH)2-22-ene-24-cyclopropyl D3、calcipotriol) 兩種佐劑藥物對於ALA-PDT之增效，以及有效治療次數。最後利用人類舌鱗狀細胞癌SCC4細胞 (human tongue squamous cell carcinoma、SCC4) 探討增效機轉。
本實驗結果顯示，在倉鼠口腔癌前病變動物模式之ALA-PDT中，光功率在700mW/cm2能量為200焦耳 (joules、J)，治療週期為72hr之持續照射治療條件時，可於病變處產生治療效果，但會對患部表面造成破壞性傷口，並有少量熱效應。若以300mW/cm2能量100J，治療週期為1週之間歇性治療條件時，可減少熱效應，但對患部的傷害仍過大。而以200mW/cm2能量100J，治療週期為1週之光照條件，則無熱效應產生，成功針對病變產生有效治療反應。
本研究發現分別以佐劑MTX及calcipotriol預處理之倉鼠口腔癌前病變模式動物，於ALA光動力診斷 (photodynamic diagnosis、PDD) 研究發現，會因不同佐劑的預處理而有不同的最佳治療窗口。控制組之ALA-PDD治療時間點為20%ALA (w/w) 水膠，於患部局部塗抹後2.5hr； MTX水膠預處理之治療時間點為6.5hr；以calcipotriol預處理之治療時間點為3.5hr。根據以上PDD條件進行ALA-PDT的結果為控制組平均需經4.8次療程，以肉眼觀察可達完全治癒 (complete response、CR) 之療效；以MTX佐劑預處理後，進行ALA-PDT達CR，平均則需經2.3次療程；以calcipotriol佐劑預處理後，進行ALA-PDT達CR平均經3.8次療程。經佐劑增效的機轉研究，初步發現SCC4細胞經不同濃度之MTX以及calcipotriol預處理後，可發現合成具光敏功能之原紫環IX (protoporphyrin IX、PpIX)上游酵素糞卟啉原氧化酶 (coproporphyrinogen oxidase、CPOX) 有上升的表現；而同為上游酵素合成PpIX的原卟啉原氧化酶 (protoporphyrinogen oxidase、PPOX) 則無明顯變化；而下游代謝PpIX之亞鐵螯合酶 (ferrochelatase、FECH) 也無明顯變化，可說明無論以MTX或calcipotriol佐劑預處理，皆可以提高細胞CPOX酵素含量，進而導致ALA-PDT的療效增加。

Photodynamic therapy (PDT) is a non-invasive cancer therapy modality. Photosensitizer or its precursor is given by topical, oral or vein-injection administration. Then photosensitizer accumulates selectively in precancerous or cancerous lesions and is then activated after irradiatiing light. The activated photosensitizer reacts with oxygen to form reactive oxygen species (ROS), resulting in tissue destruction of precancerous and cancerous tissue. Photodynamic diagnosis (PDD) of topical 5-aminolaevulinic acid (ALA) was obtained for the optimal treatment schedule to deliver optimumn PDT therapeatic results.
We established a hamster buccal pouch precancerous animal model to research on different adjuvant drugs including methotrexate (MTX) and calcipotriol for combination therapy with topical ALA-PDT in vivo. On the other hand, we used human tongue squamous cell carcinoma (SCC4) cell line, to investigate the ALA-PDT therapeutic effect with preconditioning adjuvant drugs.
For ALA-mediated PDD, the optimal treatment schedule post topical ALA administration dependeds on different kind of preconditioning adjuvant. PDD time was determined for 2.5 hr without adjuvant, 6.5 hr after 72hr MTX preconditioning, and 3.5 hr after 72hr calcipotriol preconditioning. Our study found that there are 4.8 PDT cycles for no preconditioning adjuvant, 2.3 PDT cycles post 72hr MTX preconditioning, and 3.8 PDT cycles post 72hr calcipotriol preconditioning.
In vitro, SCC4 cells were preconditioned with MTX at 0.001, 0.01, 0.1 and 1(mg/L), with calcipotriol at 10-8, 10-7, 10-6 and 10-5 M. Up-regulation of coproporphyrinogen III oxidase (CPOX), an upstream enzyme of PpIX synthesis, but there was no significant change in the regulation of protoporphyrinogen oxidase (PPOX), an enzyme co-regulating on PpIX synthesis, and ferrochelatase (FECH), the terminal enzyme in the biosynthesis of heme. Based on the above studies, it demonstrated that such combination therapy of adjuvants and ALA-PDT served a successful therapeutic modality on oral precancer therapy.

摘要 I
Abstract III
論文發表及會議報告 V
目錄 VI
圖目錄 X
表目錄 XIII
壹、 緒論 1
1-1 研究背景 1
1-2 研究動機與目的 4
貳、 實驗介紹與流程設計 6
2-1 實驗介紹 6
2-2 實驗流程設計 7
參、 文獻探討 8
3-1 光動力治療應用 8
3-1-1 人類口腔癌前病變簡介 8
3-1-2 光動力治療應用於口腔癌前病變領域 8
3-1-3 可提昇光動力治療綜效的佐劑藥物 10
3-2 理論基礎 13
3-2-1 倉鼠口腔癌前病變動物模式建立 13
3-2-2 光動力治療介紹 14
3-2-3 光動力治療三要素-光、氧氣、光感藥物 14
3-2-4 光化學反應 16
3-2-5 5-氨基酮戊酸光動力治療機轉 17
3-2-6 5-氨基酮戊酸光敏劑對癌前病變及癌症組織選擇性累積之因素 18
3-2-7 如何避免光動力引起的缺氧效應? 18
3-2-8 光動力治療所引發之免疫反應 19
3-2-9 光動力診斷 (Photodynamic diagnosis) 20
肆、 材料與方法 23
4-1 光動力治療於倉鼠口腔癌前病變之動物模式研究 23
4-1-1 本實驗用藥物與佐劑介紹 23
4-1-2 實驗藥物配製方法 23
4-1-3 口腔癌前病變之動物模式實驗方法 24
4-2 光動力治療於口腔癌前病變及變異組織之佐劑增效光動力機轉探討 26
4-2-1 動物組織之蛋白質萃取方法 26
4-2-2 BCA(bicinchoninic acid)蛋白質濃度測定與蛋白質電泳樣品製備 27
4-2-3 十二烷基硫酸鈉聚丙烯酰胺凝膠電泳（sodium dodecyl sulfate polyacrylamide gel electrophoresis、SDS-PAGE） 28
4-2-4 西方墨點法(Western Blot) 29
4-3 光動力治療於人類舌鱗狀細胞癌細胞株之佐劑增效機轉研究 30
4-3-1 細胞株 30
4-3-2 細胞解凍、繼代與冷凍方法 31
4-3-3 細胞之蛋白質萃取方法 32
4-3-4 細胞佐劑預處理以及MTT細胞存活實驗方法 33
4-3-5 統計分析 34
伍、 結果與討論 35
5-1 倉鼠口腔癌前病變與變異組織之動物模式引發 35
5-1-1 倉鼠口腔癌前病變模式組織診斷 36
5-1-2 以甲氨蝶呤塗抹劑型為光動力治療之佐劑的最佳濃度分析研究 36
5-1-3 高功率短治療週期之ALA光動力治療於倉鼠口腔癌前病變療效探討 39
5-1-4 局部ALA光動力治療之光源與治療週期評估 42
5-1-5佐劑預處理之局部ALA光動力診斷於倉鼠口腔癌前病變患部篩選最佳治 療窗口 44
5-1-5-1 以光動力診斷篩選ALA最佳治療時間 44
5-1-5-2 以光動力診斷篩選甲氨蝶呤預處理之ALA最佳治療時間 46
5-1-5-3 以光動力診斷篩選卡泊三醇預處理之ALA最佳治療時間 49
5-1-6 以發光二極體光源持續照射之佐劑預處理5-氨基酮戊酸光動力治療於倉鼠口腔癌療效探討 52
5-1-7 比較持續光照、間歇光照條件之5-氨基酮戊酸光動力治療於倉鼠口腔癌前病變療效 57
5-1-7-1 持續光照條件之5-氨基酮戊酸光動力治療於倉鼠口腔癌前病變療效 58
5-1-7-2 間歇光照條件之5-氨基酮戊酸光動力治療於倉鼠口腔癌前病變療效 60
5-1-8 高低功率、能量發光二極體光源間歇照射之佐劑預處理5-氨基酮戊酸局部光動力治療於倉鼠口腔癌前病變療效探討 61
5-1-8-1 光功率300mW/cm2、照射能量150J及間歇照射之甲氨蝶呤佐劑預處理局部ALA-PDT療效探討 61
5-1-8-2 光功率300mW/cm2、照射能量150J及間歇照射之卡泊三醇佐劑預處理局部ALA-PDT療效探討 70
5-1-8-3 光功率200mW/cm2、照射能量100J及間歇照射之甲氨蝶呤佐劑預處理局部ALA-PDT療效探討 74
5-1-8-4 光功率200mW/cm2、照射能量100J及間歇照射之卡泊三醇佐劑預處理局部ALA-PDT療效探討 82
5-1-8-5 動物實驗結果 87
5-2 增效機轉實驗結果 88
5-2-1 倉鼠口腔癌前病變動物模式佐劑增強療效機轉探討 88
5-2-2 人類舌鱗狀細胞癌細胞株佐劑增強療效機轉探討 92
5-2-3 討論 100
5-2-4 結論 108
5-2-5 未來研究方向 109
陸、 參考文獻 110
附錄A 實驗試劑配製 119
附錄B 細胞解凍、繼代培養及冷凍 121
附錄C 實驗儀器設備與藥品試劑 123

圖目次
圖2-1 實驗流程圖 7
圖3-1 倉鼠正常口腔頰囊蘇木精-伊紅染色(hematoxylin-eosin staining、H&;E staining)切片 13
圖3-2 光動力療法原理示意圖 17
圖3-3 ALA代謝路徑圖 18
圖4-1 人類舌麟狀細胞癌(Human tongue squamous cell carcinoma、SCC4) 30
圖4-2 光盤式LED光源 34
圖5-1 倉鼠口腔頰囊致癌進程 35
圖5-2 倉鼠口腔頰囊照片與蘇木精-伊紅染色(hematoxylin and eosin stain、H&;E stain)切片圖 36
圖5-3 局部ALA-PDT於倉鼠口腔癌前病變治療照片記錄 40
圖5-4 局部ALA-PDT於卡泊三醇(calcipotriol)佐劑藥物預處理之倉鼠口腔癌前病變照片記錄 40
圖5-5 局部ALA-PDT於甲氨蝶呤(methotrexate)佐劑藥物預處理之倉鼠口腔癌前病變照片記錄 41
圖5-6 局部ALA-PDT使用LumaCare鹵素燈光源或LED發光二極體光源於7天治療週期下之倉鼠口腔癌前病變病灶復原情況比較 44
圖5-7 ALA代謝PpIX之光動力診斷螢光光譜圖 45
圖5-8 ALA代謝PpIX於甲氨蝶呤(methotrexate)佐劑預處理之光動力診斷螢光光譜圖 48
圖5-9 ALA代謝PpIX於卡泊三醇(calcipotriol)佐劑預處理之光動力診斷螢光光譜圖 51
圖5-10 輕度至中度倉鼠口腔頰囊癌前病變照片與切片圖 52
圖5-11 局部ALA-PDT患部破壞及復原情況 54
圖5-12 甲氨蝶呤(methotrexate)預處理之局部ALA-PDT患部破壞及復原情況 55
圖5-13 卡泊三醇(calcipotriol)預處理之局部ALA-PDT患部破壞及復原情況 57
圖5-14 持續光照(continuous exposure)條件之局部ALA-PDT 59
圖5-15 間歇光照(fractionation exposure)條件之局部ALA-PDT 61
圖5-16 以光功率300mW/cm2間歇性光照條件進行局部ALA-PDT達150J於倉鼠口腔癌前病變治療進程 64
圖5-17 以光功率300mW/cm2間歇性光照達150J能量之局部ALA-PDT於甲氨蝶呤(methotrexate)佐劑預處理之倉鼠口腔癌前病變治療進程 67
圖5-18 以光功率300mW/cm2間歇性光照達150J能量之局部ALA-PDT於卡泊三醇(calcipotriol)佐劑預處理之倉鼠癌前病變治療進程 72
圖5-19 以光功率200mW/cm2間歇性光照達100J能量之局部ALA-PDT於倉鼠口腔癌前病變治療進程 76
圖5-20 以光功率200mW/cm2間歇性光照達100J能量之局部ALA-PDT於甲氨蝶呤(methotrexate)佐劑預處理之倉鼠癌前病變治療進程 79
圖5-21 以光功率200mW/cm2間歇性光照達100J能量之局部ALA-PDT於卡泊三醇(calcipotriol)佐劑預處理之倉鼠癌前病變治療進程 85
圖5-22 以西方墨點法檢測經甲氨蝶呤(methotrexate)預處理3天與5-氨基酮戊酸(ALA)不同時間(2.5hr及6.5hr)處理之倉鼠正常或癌前病變頰囊血紅素代謝途徑酵素CPOX、PPOX、FECH之消長變化 89
圖5-23 甲氨蝶呤(methotrexate)於正常或癌前病變倉鼠頰囊血紅素代謝途徑酵素CPOX、PPOX、FECH之蛋白質含量影響 91
圖5-24 以甲氨蝶呤(methotrexate)或二甲基亞碸(dimethyl sulfoxide)預處理人類鱗狀細胞癌SCC4細胞72hr後，施以不同濃度5-氨基酮戊酸(ALA)避光培養4hr，使用635nm紅光以持續照射(continuous lighting)或間歇照射(fractionation lighting)達10J能量之細胞存活率實驗 93
圖5- 25 以甲氨蝶呤(methotrexate)或二甲基亞碸(dimethyl sulfoxide)預處理人類鱗狀細胞癌SCC4細胞72hr後，施以不同濃度5-氨基酮戊酸(ALA)避光培養4hr，使用635nm紅光以間歇照射(fractionation lighting)達10J能量之細胞存活率實驗 94
圖5- 26 以不同濃度甲氨蝶呤(methotrexate)預處理72hr於人類舌鱗狀細胞癌SCC4之血紅素生成酵素CPOX、PPOX、FECH之蛋白質含量變化 95
圖5-27 以甲氨蝶呤(methotrexate)預處理SCC4細胞72hr後，以不同濃度5-氨基酮戊酸(ALA)避光培養4hr，再以635nm紅光間歇照射達10J能量之細胞存活率實驗 96
圖5-28 以不同濃度卡泊三醇(calcipotriol)預處理72hr於人類舌鱗狀細胞癌SCC4之血紅素生成酵素CPOX、PPOX、FECH之蛋白質含量變化 98
圖5-29 以卡泊三醇(calcipotriol)預處理SCC4細胞72hr後，以不同濃度5-氨基酮戊酸(ALA)避光培養4hr，再以635nm紅光間歇照射達10J能量之細胞存活率實驗 99

表目次
表1 不同濃度比例之SDS-PAGE配製方式 29
表2 人類舌麟狀細胞癌(Human tongue squamous cell carcinoma、SCC4)方式 31
表3 倉鼠口腔癌前病變以0.25%、0.5%及1%(w/w) MTX佐劑處理下所測得635nm光動力診斷之光子數與光子數倍數 38
表4 光動力診斷篩選局部ALA-PDT最佳治療時間 45
表5 光動力診斷篩選經甲氨蝶呤(methotrexate)佐劑預處理後，結合局部ALA-PDT之最佳治療時間 47
表6 光動力診斷篩選經卡泊三醇(calcipotriol)佐劑預處理後，結合局部ALA-PDT之最佳治療時間 50
表7 以光功率 300mW/cm2間歇性照光達150J能量之局部ALA-PDT於倉鼠口腔癌前病變患部之治療結果 65
表8 以光功率 300mW/cm2間歇性光照進行局部ALA-PDT達150J能量於甲氨蝶呤(methotrexate)佐劑預處理之倉鼠口腔癌前病變患部之治療結果 69
表9 以光功率 300mW/cm2間歇性光照達150J能量之局部ALA-PDT於卡泊三醇(calcipotriol)佐劑預處理之倉鼠口腔癌前病變患部之治療結果 73
表10 以光功率 200mW/cm2間歇性光照達100J能量之局部ALA-PDT於倉鼠口腔癌前病變患部之治療結果 77
表11 以光功率 200mW/cm2間歇性光照達100J能量之局部ALA-PDT於甲氨蝶呤(methotrexate)佐劑預處理之倉鼠口腔癌前病變患部之治療結果 81
表12 以光功率 200mW/cm2間歇性光照達100J能量之局部ALA-PDT於卡泊三醇(calcipotriol)佐劑預處理之倉鼠口腔癌前病變之治療結果 86
表13 甲氨蝶呤(methotrexate)預處理SCC4細胞72hr後之ALA-PDT細胞存活率分析 96
表14 卡泊三醇(calcipotriol)預處理SCC4細胞72hr後之ALA-PDT細胞存活率分析 99

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