建立自殺基因系統並結合介白素三號之基因療法對攝護腺癌的治療

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研究生：	游靜芳 Jing-Fang Yu
論文名稱：	建立自殺基因系統並結合介白素三號之基因療法對攝護腺癌的治療 Establishment of suicide gene system combined with interleukin-3 gene therapy for prostate cancer therapy
指導教授：	江啟勳 Chi-Shiun Chiang
口試委員:
學位類別：	碩士 Master
系所名稱：	原子科學院 - 生醫工程與環境科學系 Department of Biomedical Engineering and Environmental Sciences
論文出版年：	2006
畢業學年度：	94
語文別：	中文
論文頁數：	82
中文關鍵詞：	基因療法、介白素三號
外文關鍵詞：	Gene therapy, HSV1-TK, GCV, interleukin-3, CTL assay
相關次數：	點閱：1 下載：0
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基因療法是目前癌症治療方法中最具有潛力及前瞻性的一種新興療法。於本研究中，以TRAMP-C1攝護腺癌細胞作為研究的模式，結合自殺基因療法HSV1-tk及免疫療法IL-3此兩種療法探討治療惡性腫瘤的成效。首先，利用liposome轉殖DNA的方式建立TRAMP-C1/TK及TRAMP-C1/IL3-TK兩種腫瘤細胞株。在體外實驗結果發現，轉染後的細胞對於GCV藥物具有高度的敏感性，於低濃度時便有明顯的毒殺效果。而體內實驗中，將腫瘤細胞植入至C57BL/6J小鼠身上，施予不同的治療。結果顯示結合自殺基因HSV1-tk與細胞激素IL-3的治療後，可明顯地延緩腫瘤的生長速率，而只接受自殺基因HSV1-tk治療的組別，雖然也有延遲腫瘤生長的情形，但卻不及結合兩種治療所達到的效果。在免疫機能方面，雖然沒有如預期般的增加T細胞的毒殺能力，但在接受HSV1-tk及IL-3治療後的小鼠，其T細胞釋放IFN-γ的能力的確有大幅的提升。因此，我們建立了一個結合自殺基因治療HSV1-tk及免疫療法IL-3的系統，藉由GCV治療的毒殺效果與細胞激素IL-3刺激所提升的免疫力，兩者互相加成的作用，可以回復低落的免疫機能，增強抗腫瘤的能力，而達到延遲腫瘤生長的效果。

It is critical to develop new therapies to improve anti-tumor activity and decrease the toxicity to normal host cells. Gene therapy is a novel therapeutic approach with high potential to cure immune-resistant tumor cells. In this study, we used prostate cancer cell line, TRAMP-C1, as a research model and combined gene therapy using suicide gene system HSV1-tk/GCV and immunotherapy using murine interleukin-3 (mIL-3) to evaluate the anti-tumor effect. First, we constructed two transfected cell lines, TRAMP-C1/TK and TRAMP-C1/IL3-TK, by liposomal DNA delivery system. These transfected cell lines have high sensitivity to the cytotoxic effect of ganciclovir (GCV). The transfected tumor cells were subscutaneously inoculated into the right flank of C57BL/6J mice to establish a tumor model. The results show that the combination of HSV1-tk/GCV and mIL-3 treatment significantly delays the tumor growth rate. The treatment with HSV1-tk/GCV alone causes transient tumor growth delay in comparison to combined therapy. In immune functional assay, we didn’t observe expected enhancement of cytotoxic T lymphocyte response. However, the ability of CD4 T cells secreting IFN-γ is improved in combined treatment with HSV1-tk/GCV and mIL-3. These results indicate that this combined system can recover down-regulated immunity and enhance anti-tumor activity. In summary, we have already established a combined gene therapy system, which relies on the cytotoxicity of GCV treatment and immune responses stimulated by mIL-3. This system can active CD4 T cells to secret IFN-γand delay the tumor growth rate.

英文摘要 ………………………………………………………………Ⅰ
中文摘要 ………………………………………………………………Ⅲ
誌謝 ……………………………………………………………………Ⅳ
目錄 ……………………………………………………………………Ⅴ
第一章 序論 ……………………………………………………………1
1 攝護腺癌 .......................................................................................1
1.1  攝護腺癌簡介 ……………………………………………1
1.2  攝護腺癌的診斷 …………………………………………1
1.3  傳統治療方法 ……………………....................................2
2 基因治療 …………………….......................................................4
3 第一型單純疱疹病毒胸腺嘧啶激脢/丙氧鳥苷系統 ..................6
3.1   HSV1-tk/GCV的歷史 .....................................................6
3.2   HSV1-tk/GCV的作用機轉 .............................................7
3.3   旁觀者效應 ......................................................................7
3.4   HSV1-tk/GCV的應用 .....................................................8
4 介白素3號 .................................................................................8
4.1   介白素3號在免疫系統中的功能與角色 ......................8
4.2   介白素3號對腫瘤的療效 ..............................................9
4.3   介白素3號的副作用 ....................................................10
5 研究目的與內容 .......................................................................11
第二章 材料與方法 ..............................................................................12
1質體的構築 .................................................................................12
1.1   聚合酶素連鎖反應 ........................................................12
1.2   限制酶酵素切割反應 ....................................................13
1.3   限制酶酵素切割反應 ....................................................14
1.4   DNA片段的純化 ...........................................................14
1.5   DNA的接合作用 ...........................................................15
1.6   細菌熱休克轉殖法 ........................................................15
1.7   微量純化製備質體 ........................................................16
1.8   洋菜膠電泳 ....................................................................16
1.9   中量純化製備質體 ........................................................17
2 細胞培養 ...................................................................................18
2.1   配製DMEM細胞培養液 ..............................................18
2.2   細胞培養液的配方 ........................................................18
2.3   細胞繼代 ........................................................................19
3 質體在轉染細胞中的表現 .......................................................19
3.1   細胞的轉染 ....................................................................19
3.2   RNA的製備 ...................................................................20
3.3   RT-PCR ............................................................................21
3.4   酵素連結免疫吸附法(ELISA) .......................................23
3.6   細胞對於GCV的敏感性試驗 ......................................24
3.7   細胞存活試驗(MTT assay) ............................................24
4 動物實驗 ...................................................................................25
4.1   動物來源 ........................................................................25
4.2   動物分組 ........................................................................25
4.3   植入TRAMP-C1、TRAMP-C1/IL3-TK、TRAMP-C1/TK
            腫瘤細胞株 ....................................................................26
4.4   血清樣本 ........................................................................26
4.5   酵素連結免疫吸附法 ....................................................27
4.6   RNA的製備 ...................................................................27
4.7   RT-PCR ............................................................................27
4.8   注射GCV ........................................................................27
4.9   腫瘤生長曲線 ................................................................28
5 免疫機能測試 ...........................................................................28
5.1   純化脾臟T細胞 ............................................................28
5.2   酵素連結免疫吸附法 ....................................................29
5.3   細胞毒殺測試 ................................................................29
第三章 實驗結果 ..................................................................................31
1質體的構築 .................................................................................31
1.1   IRES-IL3-TK質體的構築 .............................................31
1.2   IRES-TK質體的構築 ....................................................32
2 構築好的質體DNA在轉染細胞中的表現 .............................33
2.1   將構築好的質體DNA送入細胞中 ..............................33
2.2   轉染細胞中基因mRNA的表現情形 ............................34
2.3   轉染細胞中蛋白質的表現情形 ....................................34
2.4   轉染細胞對於GCV藥物的敏感性實驗 ......................35
3 轉染細胞在動物體內的反應 ...................................................36
3.1   腫瘤細胞中mRNA的表現情形 ....................................36
3.2   血清中細胞激素IL-3的分析 ........................................37
3.3   腫瘤生長曲線 ................................................................38
4  T細胞免疫反應測試 ..............................................................39
4.1   純化T細胞 .....................................................................39
4.2   檢視T細胞表現IFN-γ與IL-4的情形 ........................39
4.3   檢測T細胞毒殺能力 .....................................................40
第四章 討論 ..........................................................................................42
圖表說明 ................................................................................................50
圖表 ........................................................................................................56
附錄一 ....................................................................................................73
附錄二 ....................................................................................................74
附錄三 ....................................................................................................75
參考文獻 ................................................................................................77

                                

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