臺灣博碩士論文加值系統

開發中可即時評估化療療效的光纖感測平台於本研究中被用於皮膚癌化療成效評估。它藉由微侵入方式即時檢測活體內腫瘤經藥物治療後誘發癌細胞凋亡情形，經由回傳訊號立即調整化療劑量或劑型以達成個人化治療目標。先前此平台已證實可用於檢測乳癌經化療藥物誘發細胞凋亡情形，為了證明此個人化光纖感測平台並非只侷限於單一癌症個人化治療應用，因此本研究將此系統應用於皮膚癌化療成效評估。研究內容主要分為五個部份探討並以人類惡性黑色素瘤細胞株 A375.S2做為研究對象。第一部份以存活度探討化療藥物cisplatin誘發細胞凋亡最適濃度，從分析結果選定18小時濃度25.2μM下為最佳誘發細胞凋亡濃度；第二及第三部份分別使用流式細胞儀與螢光顯微鏡確認此時間點與濃度是否能有效造成細胞凋亡，流式細胞儀分析結果顯示，化療誘發凋亡組別早期凋亡細胞比例由8.7%大幅上升至23.9%；螢光顯微鏡觀測下，化療藥物誘發細胞凋亡下高螢光強度細胞個數明顯比控制組多，因此證實此時間點與濃度確實可以有效造成細胞凋亡。第四部份探討解決立體細胞空間分佈的兩種螢光試劑 (FM1-43和Mb) 是否如理論上的表現，從結果證實細胞凋亡指示劑FM1-43 隨著濃度增加到最佳誘發細胞凋亡濃度時螢光強度增加了37.5%，而細胞空間分佈指示劑Mb並沒有因為細胞凋亡而產生螢光上的變化；第五部份探討將其應用於腫瘤內可行性，因此以仿生膠體模擬腫瘤內部並以光纖感測系統進行檢測，從結果證實FM1-43螢光動態變化差值與細胞密度以及細胞凋亡程度均呈正相關，而Mb螢光動態變化差值以統計學來說並無顯著差異；除此之外以兩者螢光物比值確認此光纖感測平台之靈敏區域，並以此區域建立基準閥值判斷A375.S2細胞是否有無細胞凋亡情形。最後，經由上述研究初步證明此套光纖感測平台確實能有效檢測cisplatin誘發A375.S2細胞凋亡，而在未來將繼續探討高密度細胞狀態中螢光動態變化趨勢以及進一步於動物模型中進行活體內檢測，並期望能與主動促進控制釋放平台與光纖感測平台做結合達到診斷與治療之功能。

A developing fiber-optical biosensing platform for real-time evaluation of chemotherapy efficacy was investigated for skin cancer management. The minimal invasive system can detect the apoptosis of cancer cells induced by chemotherapy, and immediately adjust the administrating chemotherapy dosage according to the detected signal. The system was verified previously in detecting breast cancer cells apoptosis induced by chemotherapy. In order to demonstrating the system with capabilities in managing multiple cancers, we provided proof of principle by demonstrating the in vivo drug-induced skin cancer apoptosis sensing capability of the fiber-optic platform.
Human melanoma cell line A375.S2 was chosen in the study, which was divided into five parts. Firstly, we explored the optimal concentration of cisplatin, a conventional chemotherapy agent for melanoma to induce apoptosis by cell viability assay. The results indicated that melanoma cell co-cultured 18 hours with 25.2μM cisplatin resulting in sufficient apoptosis. In 2nd and 3nd parts of the study, flow cytometry analysis and fluorescence microscopy inspection were applied to confirm the apoptosis of melanoma cell induced by cisplatin. Flow cytometry analysis showed that the proportion of early apoptotic cells in chemotherapy-induced apoptosis group increased significantly from 8.7% to 23.9%. From the fluorescence microscope images, cells with higher fluorescence intensity were increased significantly in chemotherapy treated group. In 4th part of study, we evaluated if the two fluorescence reagents (FM1-43 and Mb) ideal for the task of apoptosis sensing. The results show that the fluorescence intensity of apoptosis indicator, FM1-43, increased 37.5% when melanoma exposed to cisplatin, but spatial distribution indicator, Mb, did not have significant fluorescence changes after cisplatin treatment. In 5th part of study, the system was applied to detect cisplatin induced apoptosis of melanoma cells in polyethylene glycol hydrogel, simulating the 3-dimension distribution, for verifying the capability of the system in detecting the in vivo apoptosis. The results indicated that the dynamic difference of FM1-43 fluorescence was directly correlated with cell density and the extent of apoptosis, but Mb was not statistically significant. In conclusion, we proved that this fiber-sensing platform could effectively detect apoptosis of A375.S2 cells induced by cisplatin. Advantages of the tunable chemotherapy delivery and immediate efficacy evaluation of the fiber-optic system in skin cancer management will be further evaluate in the following study.

中文摘要 i
Abstract ii
目錄 iv
圖目錄 vi
表目錄 viii
第一章 緒論 1
1.1 研究動機 1
1.2 研究目的 2
第二章 文獻探討 4
2.1 以光纖感測平台做為乳癌個人化治療工具 4
2.1.1 藉由光纖系統檢測活體內腫瘤經藥物誘發後細胞凋亡 4
2.1.2 藉由光纖系統即時調整定點藥物劑量 8
2.2 皮膚癌介紹 11
2.2.1 皮膚癌的種類和表徵 12
2.2.2 皮膚癌的診斷 13
2.2.3 皮膚癌的治療 14
第三章 實驗材料與方法 15
3.1 實驗儀器設備 15
3.2 實驗藥品材料 16
3.3 藥品配置 17
3.3.1 細胞培養液 17
3.3.2 磷酸生理緩衝溶液 (Phosphate Buffer Solution, PBS) 17
3.4 細胞培養 18
3.4.1 解凍細胞 18
3.4.2 細胞培養 (Cell Culture) 18
3.4.3 細胞繼代 (Cell Passage) 18
3.4.4 細胞凍存 18
3.5 實驗方法 19
3.5.1 皮膚癌化療藥物選擇 19
3.5.2 細胞凋亡指示劑選擇 20
3.5.3 細胞空間指示劑選擇 21
3.5.4 細胞存活率測試 22
3.5.5 追蹤螢光染劑於Phosphatidylserine (PS) 累積 22
3.5.6 流式細胞儀技術 23
3.5.7 細胞凋亡檢測 27
3.5.8 細胞吸附螢光檢測 27
3.5.9 光纖感測平台 28
3.5.10 仿生立體細胞分佈光纖感測 29
第四章 實驗結果與討論 30
4.1 以細胞存活度探討cisplatin誘發細胞凋亡最適濃度 30
4.2 以流式細胞儀及螢光顯微鏡確認cisplatin誘發凋亡之情形 31
4.3 以螢光光譜儀定量螢光物吸附與細胞凋亡的關聯性 34
4.4 仿生立體3D分佈系統中cisplatin誘發細胞凋亡與螢光物吸附動態變化趨勢 36
第五章 結論與未來展望 39
第六章 參考文獻 40

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