臺灣博碩士論文加值系統

一、 膀胱癌之基因治療
目前對於膀胱癌的治療, 通常是在外科手術後施以化學治療或放射線治療, 但病患治療後仍有相當高的復發率。 是以尋求新的輔助性療法來提高膀胱癌的治癒率, 實為重要的工作。 而近來發展快速且範圍廣泛的基因治療(gene therapy)觀念, 便成為我們研究的方向。
本實驗室以C3H/HeNCrj 品系的小鼠及其膀胱癌細胞株MBT-2作為研究模式。 先前將已利用反轉錄病毒分別送入細胞素基因(cytokine) Interleukin-2, Interleukin-4, granulocyte-macrophage colony-stimulating factor的癌細胞(MBT-2-IL-2, MBT-2-IL-4, MBT-2-GM-CSF) 經過放射線照射後, 作為腫瘤疫苗(tumor vaccine)來進行治療, 發現此三種腫瘤疫苗皆有相當療效。 其後更利用MBT-2細胞有致癌基因neu過度表現的特性, 將可以表現大鼠(rat) neu的質體pSV-neu注入小鼠的肌肉中, 當作DNA 疫苗(DNA vaccine), 來合併腫瘤疫苗MBT-2-IL-2使用。 結果發現可使腫瘤發生率降低到22 %。
本次實驗我們利用另一腫瘤疫苗MBT-2-GM-CSF, 合併DNA 疫苗(pSV-neu)來觀察是否也具有同樣的治療效果。 並更進一步構築片段的neu (truncated neu)─ N端的neu (pRc/CMV-N'-neu) 及 C端的neu (pRc/CMV-C'-neu)─ 作為合併使用的DNA疫苗。
由這次實驗的結果顯示, 合併腫瘤疫苗MBT-2-GM-CSF與DNA 疫苗(pSV-neu或pRc/CMV-N'-neu)在腫瘤生成率方面(83.3% 及100%)與對照組(76.4%)比較, 並無明顯治療效果。 不過在藉由西方點墨法(Western blotting)來偵測老鼠體內是否有產生anti-neu抗體的試驗中, 發現在合併處理腫瘤疫苗MBT-2-GM-CSF及DNA疫苗pRc/CMV-N'-neu的老鼠有anti-neu的抗體產生; 另一方面, 在將老鼠之腫瘤作冷凍切片免疫染色(immunohistochemistry) 來觀察何種淋巴球(lymphocyte)滲入(infiltration)腫瘤組織較多的試驗中, 發現在處理腫瘤疫苗、 DNA疫苗或合併處理後的老鼠, 其腫瘤組織中輔助T細胞(helper T cells, CD4)都較對照組為多, 而殺手T細胞(cytotoxic T cells, CD8)則無增加的現象。
由這些結果可以推論腫瘤疫苗MBT-2-GM-CSF與DNA 疫苗(pSV-neu或pRc/CMV-N'-neu)的確活化了老鼠體內特定的免疫反應, 但卻不足以抑制腫瘤生長。
二、 膀胱癌抗藥性之研究
化學療法是治療癌症的一種有效且重要的方法之一, 但是隨著長期的化療, 癌細胞很容易就產生抗藥性而使得治療效果不彰。 癌細胞對抗癌藥物產生抗性牽涉到許多細胞機制, 在過去的研究認為可能與細胞內一些蛋白表現的消長有關。
之前本實驗室曾分析實驗室既有之九株人類膀胱癌細胞, 其可能與抗藥性機制有關之蛋白質的表現量與對adriamycin, cisplatin, melphalan, methotrexate等抗癌藥物產生抗性之相關性。 結果發現p16表現量與細胞對adriamycin, cisplatin, melphalan 產生抗性具有高度相關性, 於是我們想研究p16在細胞產生抗藥性中所扮演的角色。
我們將p16 之cDNA(sense or anti-sense)構築在可藉由IPTG誘導的系統(IPTG inducible system), 而後將順意的(sense)p16轉染(transfection)到p16表現量低的細胞株ScaBER; 反意的(anti-sense) p16則轉染到p16表現量高的細胞株TCC-SUP中, 藉藥物G418的篩選來挑選穩定的轉染細胞株(stable transfectant)。 結果我們僅挑選到能夠藉由IPTG誘導來降低p16表現量的轉染細胞株1201-1。 藉IPTG誘導轉染細胞株1201-1所作的藥物毒殺曲線中顯示, 降低p16的表現量會使得1201-1細胞對Cisplatin較為敏感。

I. Gene Therapy in Bladder Cancer
Combination of surgery, chemotherapy and radiotherapy is the standard therapy for invasive bladder cancer, but the five-year recurrence rate is still discouraging (>50%). Therefore, it is important to develop other effective therapies and gene therapy is a potential candidate.
Our laboratory has established 3 tumor vaccines, MBT-2-IL-2, MBT-2-IL-4, MBT-2-GM-CSF for MBT-2 cells and C3H/HeNCrj mice animal model. Previous study indicated that MBT-2-IL-2 provide better therapeutic efficacy: tumor formation rate of mice ? 60 % when treating alone, ? 22 % when combining with DNA vaccine pSV-neu encoding rat oncogene neu.
In this study, we evaluated the therapeutic efficacy of combination of tumor vaccine MBT-2-GM-CSF and DNA vaccines which include full-length neu(pSV-neu) and truncated neu (pRc/CMV-N'-neu, pRc/CMV-C'-neu).
The immunohistochemistry studies showed that significant infiltration of CD4+ T helper cells in the tumor of the mice treated with tumor vaccine, DNA vaccine, or both. On the other hand, we detected anti-neu antibody in the serum of the mice that were treated with combination of tumor vaccine MBT-2-GM-CSF and DNA vaccine pRc/CMV-N'-neu. However, the tumor formation rate was no difference between untreated mice( 76.4 %) and combination-therapy mice(83.3 % and 100 %).
These results suggest that tumor vaccine MBT-2-GM-CSF or DNA vaccines actually activate certain immune response, but that is insufficient to repress tumor growth.
II. Drug Resistance in Bladder Cancer Cell
Development of drug resistance is an important problem in treating bladder cancer. Our laboratory have determined the expression of cell-cycle related genes in 9 bladder cancer cell lines, and correlated with the drug resistance to many chemotherapeutic drugs, and we discovered that the expression of p16 is correlated to drug resistance.
To further investigate what the role of p16 is in drug resistance, the p16 and antisense p16 cDNA was constructed into an IPTG-inducible system. We delivered IPTG-inducible-p16 into ScaBER cell line that has low expression of p16, and IPTG-inducible-antisense-p16 into TCC-SUP, which has high expression of p16.
We did not obtain any transfectant on ScaBER cell that was possible due to the cell cycle arrest by leakage-expression of p16. On the other hand, we established a stable transfectant 1201-1. Addition of IPTG in this transfectant can downregulate the expression of p16. The drug resistance of this cell line to Cisplatin and other drugs was tested in the presence and absence of IPTG. The sensitivity to Cisplatin indeed changed by the addition of IPTG.

授權書
口試合格證明書
摘要 I
中文摘要 I
英文摘要 IV
誌謝 VI
目錄 VIII
表目錄 XIII
圖目錄 XIV
縮寫表 1
緒論 4
一、 膀胱癌之基因治療 4
二、 膀胱癌抗藥性之研究 10
材料與方法 15
一、 膀胱癌之基因治療 15
(一) 細胞培養
A. 材料
1. 細胞株
2. 試藥
3. 溶液配製 16
B. 方法 17
1. 解凍細胞 17
2. 例行工作 18
3. 繼代培養
4. 凍細胞 19
5. 計數細胞
(二) 質體製備 20
A. 材料
1. 試藥 21
2. Maker
3. Kits 22
4. 溶液配製
B. 方法 23
1. 少量質體製備(WizardR Plus Minipreps DNA purification system)
2. 大量質體製備(QIAGEN plasmid Mega kit) 24
(三) 構築表現N端neu的質體pRc/CMV-N'-neu 26
A. 材料
1. 質體
2. 聚合?連鎖反應引子(primer)
3. 試藥 27
4. 酵素
5. Kits
6. 製備勝任細胞之菌株 28
7. 溶液配製
B. 方法 29
1. 質體製備
2. 聚合?連鎖反應(PCR)
3. 酒精沉澱 30
4. 限制?水解 31
5. 瓊脂醣膠(agarose gel)上回收DNA 32
6. 接合?反應 33
7. 勝任細胞之製備
8. 轉形作用(transformation) 34
(四) 去除內毒素(endotoxin) 35
A. 材料
1. 試藥
2. 溶液配製
B. 方法
(五) 動物實驗 36
A. 材料 36
1. 細胞株
2. 實驗動物
B. 方法
1. 腫瘤細胞之接種 37
2. 腫瘤疫苗之製備及使用
3. DNA疫苗之製備及使用
(六) 治療效果評估方式 A. 材料
1. 細胞株
2. 試藥 38
3. Kits 39
4. 抗體
5. 溶液配製 40
B. 方法 44
1. 腫瘤生成率
2. 腫瘤大小
3. 西方點墨法偵測老鼠血清 44
4. 腫瘤冷凍切片免疫染色 47
二、 膀胱癌抗藥性之研究 49
(一) 細胞培養
(二) 構築表現p16INK4a的質體pLacM-p16
A. 材料
1. 質體
2. 聚合?連鎖反應引子(primer)
3. 試藥 50
4. 酵素
5. Kits
6. 製備勝任細胞
7. 溶液配製
B. 方法
1. 質體製備
2. 聚合?連鎖反應 51
3. 酒精沉澱 52
4. 限制?水解
5. 瓊脂醣膠(agarose gel)上回收DNA 53
6. 接合?反應 53
7. 勝任細胞之製備
8. 轉形作用(transformation)
(三) 建立轉殖細胞株(stable transfection)
A. 材料
1. 細胞株
2. 質體 54
3. 試藥
4. 溶液配製
B. 方法 55
1. 質體製備
2. 轉染細胞
(四) 西方墨點法確認轉染成功之轉殖細胞株(stable transfectant) 56
A. 材料
1. 細胞株
2. 相關之試藥、Kits、溶液配製
3. 抗體
B. 方法 57
1. IPTG 誘導基因表現
2. 製備細胞萃取液(cell lysate)
3. 以SDS-PAGE跑電泳
4. 抗體辨識標的蛋白
(五) 藥物毒殺細胞曲線 58
A. 材料
1. 試藥
2. 溶液配製
B. 方法 59
1. MTT 試驗
2. Trypan Blue排除法 60
結果 61
一、 膀胱癌之基因治療 61
二、 膀胱癌抗藥性之研究 65
討論 68
一、 膀胱癌之基因治療 68
二、 膀胱癌抗藥性之研究 71
參考文獻 74
表 A~B
圖 i~xviii
自述 117

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