臺灣博碩士論文加值系統

環磷醯胺 (Cyclophosphamide, CTX)，中文藥名為癌德星 (Endoxan®) ，是一和氮芥化學相關的合成烷化劑，具有抗腫瘤及免疫抑制的活性，在使用低藥劑的環磷醯胺 (Cyclophosphamide) 期間會有輕微的淋巴細胞清除 (lymphodepletion) 現象，在免疫系統回復的期間給予抗原會有效地增加免疫治療 (immunotherapy) 的效果。
本實驗室將癌細胞以慢病毒 (lentivirus) 轉型成GVAX疫苗，經序列優化 (codon-optimized) 過的巨噬細胞聚落刺激因子 (granulocyte-macrophage colony-stimulating factor, GM-CSF) 可增加GM-CSF的產量，使免疫作用加強，但是在腫瘤體積約200立方毫米的老鼠模型上治療效果不佳，為了解決此問題，因此選用cyclophosphamide化療用藥及GVAX疫苗搭配治療TC-1腫瘤小鼠模式，並找出適當施打的時間點進行治療，及施打GVAX疫苗前給藥的時間。
在資料數據整理過後，GVAX疫苗會促使腫瘤生長，在加入CTX治療過後腫瘤生長速度減緩，但是還是無法完全治癒腫瘤，其中我們發現單獨給予CTX的治療結果比預期的結果更佳，而CTX與GVAX合併治療的效果在引發免疫反應上有顯著的效果。

We use low-dose Cyclophosphamide (Endoxan®, CTX), a synthetic alkylating agent chemically related to the nitrogen mustards with antineoplastic and immunosuppressive activities, to make immune system lightly lymphodepletion. It initiates a good response on tumor flattening after we apply low-dose CTX to TC-1 tumor-bearing mice model, and also enhanced the effect of immunotherapy.
Our previous study suggested that GVAX vaccine, which transduced a fragment of codon-optimized GM-CSF by lentivirus, would enhance GM-CSF secretion. The efficacy did not achieve our expectation when the tumor size was about 200 mm3. To overcome the problem, we choose cyclophosphamide to treat TC-1 tumor-bearing murine model with GVAX vaccine to find out the best timing to apply cyclophosphamide before the GVAX vaccine was given.
After analyzing the GVAX treatment data, we found that the GVAX vaccine could facilitate tumor growth. We combined cyclophosphamide and GVAX to increase the efficacy of curing the tumor. After CTX treatment tumor growth slow down but tumor still could not be cured. CTX-only treatment seemed to the best effect in these treatment and CTX-GVAX combination treatment can induce immune responses significantly.

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致謝 iii
中文摘要 iv
Abstract v
目錄 vi
圖目錄 ix
第一章：緒論 - 1 -
1.1 人類乳突病毒(HPVs) - 1 -
1.1.1子宮頸癌與人類乳突病毒 - 1 -
1.1.2 HPV病毒致癌基因 - 2 -
1.1.3 人類乳突病毒疫苗 - 3 -
1.2 環磷醯胺 (Cyclophosphamide, CTX) - 4 -
1.3 顆粒球巨噬細胞群落刺激因子(granulocyte-macrophage colony-stimulating factor, GM-CSF) - 5 -
1.4 調節T細胞及腫瘤 - 7 -
1.4.1 調節T細胞(Regulatory T cell, Treg cell) - 7 -
1.4.2 Treg的抑制機制 - 7 -
1.5 實驗目的 - 8 -
第二章：材料方法 - 9 -
2.1 DNA 重組 - 9 -
2.2 細胞株 - 9 -
2.3 小鼠 - 10 -
2.4 藥劑 - 10 -
2.5 動物實驗 - 10 -
2.6 酵素免疫分析 (Enzyme-linked immunoSorbent assay, ELISA) - 11 -
2.7 淋巴球再刺激 - 12 -
2.8 全血染色分析 - 13 -
2.9 流式細胞儀分析 - 13 -
第三章：結果 - 15 -
3.1 TC-1/cGM穩定地大量表現GM-CSF - 15 -
3.2 建立穩定的腫瘤模式 - 15 -
3.3 決定給予每隻小鼠2 mg CTX作為此實驗之劑量 - 15 -
3.4 只給予CTX的單獨治療比CTX與GVAX合併治療對於腫瘤生長的趨勢無法改善 - 15 -
3.5 CTX與GVAX合併治療可以產生腫瘤免疫反應 - 16 -
3.6 在脾臟與淋巴結中Treg並沒有太多比例上的變化 - 17 -
3.7 在全血中Treg細胞並沒有明顯的變化 - 17 -
第四章：結論 - 18 -
參考資料 - 20 -

 
圖目錄
圖 一 TC-1/CGM可以穩定表現GM-CSF - 23 -
圖 二 腫瘤模式建立 - 24 -
圖 三 2 MG CTX的使用劑量會使腫瘤明顯的延緩生長 - 25 -
圖 四 在第一輪的實驗合併治療有延緩腫瘤的生長趨勢 - 26 -
圖 五 第四次實驗只給予CTX治療的組別大部分腫瘤消失 - 27 -
圖 六 第五次實驗只給予CTX治療的組別大部分腫瘤消失 - 28 -
圖 七 在第一、四、五次實驗中只要有給予CTX生存率較好 - 29 -
圖 八 CTX及GVAX合併治療可以增加CD4及CD8 T細胞免疫反應 - 30 -
圖 九 CTX及GVAX合併治療可以增加TH2免疫反應 - 31 -
圖 十 TREG細胞在脾臟及淋巴結所佔的T細胞比例 - 32 -
圖 十一 全血分析 - 33 -

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