臺灣博碩士論文加值系統

順式-二氨二氯合鉑 (cis-Diamineplatinu (Ⅱ) dichloride, CDDP) 為目前最常見的化學治療藥物，主要被應用在治療如頭頸癌、泌尿道癌以及肺癌等其他類型癌症。雖然 CDDP 在臨床的運用上十分廣泛，然而其本身的副作用反造成臨床醫師在治療病患上面臨諸多困難，最為顯著已知副作用包括：耳毒性、腎毒性、神經毒性以及噁心嘔吐等。在本研究中主要，設計可包覆 CDDP 於白蛋白超音波微氣泡對比劑 (Microbubbles, MBs)，一種穩定且可承載足夠劑量抗癌藥物之新型白蛋白超音波微氣泡對比劑，並結合超音波產生的穴蝕效應打開細胞膜通透度，增加腫瘤內局部的藥物累積濃度，抑制下咽癌癌細胞生長，減少周圍組織的傷害及副作用。
CDDP-MBs 透過動態光散射粒徑分析儀量測粒徑及庫爾特計數器分析濃度等實驗，並分別進行細胞實驗 (n=5) 與動物活體實驗 (n=7) 其實驗組別分為 (1)控制組 (C)；(2) 單純白蛋白微氣泡對比劑 (MBs)；(3) CDDP溶液 (CDDP)；(4) CDDP溶液混合MBs (CDDP+MBs)；(5) CDDP包覆於MBs (CDDP-MBs)；(6) CDDP混合 MBs 結合超音波 (CDDP+MBs+US)； (7) CDDP 包覆於 MBs 結合超音波 (CDDP-MBs+US)。
本研究以不同濃度之白蛋白溶液與 1.2 mg/ml CDDP 所配製而成的 CDDP-MBs，其粒徑大小隨白蛋白濃度132 mg/ml、140 mg/ml、150 mg/ml的增加而改變，故粒徑大小依序為 1.70 ± 0.73、2.42 ± 0.61、2.44 ± 4.49 µm；其中以白蛋白濃度 140 mg/ml包覆 CDDP 所製成的微氣泡對比劑數目含量最高；包覆效率達 10.09 ±
0.23 %；在體外試驗中，未經超音波施打前，CDDP-MBs 組對下咽癌細胞毒性效果不佳，但經超音波施打後整體毒殺能力大幅提升，平均細胞存活率下降 26 % (p<0.001)；在動物試驗中，可觀察到 CDDP-MBs+US 與 CDDP 治療趨勢結果相似，但 MBs 包覆 CDDP 時可使其副作用減低，從腎臟病理切片結果觀察到 CDDO+MBs+US 比CDDP-MBs+US有較嚴重的損傷，故綜合以上 CDDP-MBs 其治療的效果能降低 CDDP 所引起的副作用。

Cisplatin (cis-Diamineplatinu (Ⅱ) dichloride, CDDP) is one of the most frequently used agents in the treatment a variety of tumor, particularity head and neck, genitourinary, and lung cancer. Although CDDP was widely used in clinical, the side effects (Ototoxicity, Nephrotoxicity, Neurotoxicity, Nausea and Vomiting) limit the dose of cisplatin that can be safely administered.
The aim of this study is to establish a new, stable, high drug-loading capacity albumin-shelled microbubbles (MBs) combined with ultrasound (US) to enhance hypopharyngeal cancer treatment. The CDDP-MBs combined with ultrasound can increased the capillary permeability through cavitation mechanism. It increased the local concentrations of tumor site, inhibited the hypopharyngeal cancer cell growth without causing surrounding normal tissues damage and reduced the side effects.
The size distribution and concentration of the CDDP-loaded MBs in suspension were measured by DLS and Coulter Counter respectively. The in vitro (n=6) and in vivo (n=7) experimental parameters will be divided into eight groups: (1) no treatment (Control); (2) microbubbles (MBs) alone; (3) cisplatin solution (CDDP) alone; (4) CDDP solution mixed with MBs (CDDP+MBs); (5) CDDP-loaded MBs (CDDP-MBs) alone; (6) US combined with penetrating CDDP (CDDP+US); (7) US combined with MBs and penetrating CDDP (CDDP+MBs+US); (8) US combined with CDDP-MBs (CDDP-MBs+US).
In our research, the CDDP-MBs prepared with different concentrations of albumin solution (132 mg/ml, 140 mg/ml and 150 mg/ml) and 1.2 mg/ml CDDP solution. The mean diameters of CDDP-MBs with various concentrations of albumin were 1.70 ± 0.73, 2.42 ± 0.61, 2.44 ± 4.49 µm respectively. The maximum loading efficiency of CDDP on MBs (with 140 mg/ml albumin : 1.2 mg/ml CDDP) was 10.09 ± 0.23 %. In the in vitro study, CDDP-MBs can increase 26 % cell toxicity after ultrasound irradiation (p<0.001). In small animal treatment, the treatment efficacies in CDDP-MBs+US and CDDP groups were similar before 17 days. Instead of CDDP, it can be expected that CDDP-loaded microbubbles reduces the side effect more significant.

中文摘要 i
ABSTRACT ii
誌謝 iii
目錄 vi
圖目錄 x
表目錄 xii
第1章 緒論 1
1.1 癌症 1
1.1.1 頭頸癌 1
1.1.2 下咽癌 2
1.2 癌症的治療發展 4
1.2.1 手術 4
1.2.2 放射線 5
1.2.3 化學治療 5
1.2.4 標靶治療 5
1.2.5 超音波 6
1.3 鉑金類藥物簡介 7
1.3.1 順式-二氯二氨合鉑發展 7
1.3.2 藥理作用機制 8
1.3.3 副作用 9
1.4 人類血清白蛋白 10
1.4.1 人類血清白蛋白作為載體相關之研究 11
1.5 人類血清白蛋白與順式-二氯二氨合鉑 (Albumin-CDDP Complex) 12
1.5.1 結合機制 13
1.5.2 臨床藥理應用 13
1.6 微氣泡對比劑 14
1.6.1 超音波微氣泡對比劑 14
1.7 超音波結合微氣泡對比劑增加局部治療 16
1.7.1 超音波簡介 16
1.7.2 藥物傳輸機制 17
1.7.3 穴蝕效應 19
1.8 研究動機 22
第2章 材料與方法 23
2.1 研究架構 23
2.2 藥品與設備 24
2.2.1 藥品 24
2.2.2 設備 24
2.3 無菌人類血清白蛋白包覆順式-二氯二氨合鉑之製作 26
2.3.1 白蛋白微氣泡對比劑之物理性質分析 26
2.3.1.1HPLC定性分析 26
2.3.1.2光學定性分析 29
2.3.1.3粒徑分析 29
2.3.1.4濃度分析 30
2.3.1.5雙束型聚焦離子束顯微鏡拍攝 31
2.4 白蛋白微氣泡對比劑之白蛋白含量測量 31
2.5 白蛋白球殼包覆藥物之定量分析 31
2.6 高頻超音波動物影像系統模擬打破效率 32
2.6.1 24 well內之打破效率 32
2.6.2 仿體內模擬動物體內之打破效率 33
2.7 體外細胞毒殺實驗 35
2.7.1 細胞株及繼代培養 35
2.7.2 細胞計數 35
2.7.3 培養液配製 36
2.7.4 凍細胞及解凍細胞 36
2.7.5 細胞實驗設計 37
2.4.5.1 細胞存活率分析 37
2.4.5.2 半抑制濃度 38
2.4.5.3 細胞治療分組 38
2.4.5.4 實驗方法 39
2.8 體內異種移殖抗腫瘤實驗 41
2.8.1 動物品系及飼養條件 41
2.8.2 異種移殖 41
2.8.3 抗腫瘤實驗分組與療程 41
2.8.4 3D非侵入式活體分子影像系統 44
2.8.5 採血及病理組織切片 44
2.9 統計分析 44
第3章 實驗結果 45
3.1 微氣泡物理性質 45
3.1.1 無菌微氣泡對比劑 45
3.1.2 HPLC 定性分析 45
3.1.3 濃度及粒徑分析 48
3.1.4 ICP-MS定量分析 50
3.1.5 光學定性影像 51
3.1.6 雙束型聚焦離子束顯微鏡表面觀察 52
3.2 高頻超音波影像分析超音波能量打破效率之參數評估 52
3.2.1 24 well內模擬體外實驗 52
3.2.2 細胞體外試驗於不同超音波對於細胞的影響 54
3.2.3 仿體模擬體內實驗 54
3.3 體外細胞毒殺試驗 56
3.3.1 CDDP針對 FaDu Cell IC50 測量結果 56
3.3.2 細胞毒性分析 57
3.3.3 細胞外觀觀察 59
3.4 體內異種移植抗腫瘤實驗 60
3.4.1 活體內冷光影像及腫瘤生長趨勢之結果 60
3.4.2 組織切片 62
第4章 討論 64
第5章 結論 69
參考文獻 70

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