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研究生:林俞宏
研究生(外文):Yo-Hon, Lin
論文名稱:探討干擾素beta在促進樹圖狀細胞所誘發之毒殺性T細胞於小鼠大腸直腸癌免疫治療之效果
論文名稱(外文):Effective induction of anti-colorectal carcinoma cytotoxic T cell by co-administration of Interferon beta vector and dendritic cells
指導教授:吳立真
指導教授(外文):Li-Chen, Wu
學位類別:碩士
校院名稱:國立暨南國際大學
系所名稱:生物醫學科技研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:119
中文關鍵詞:beta型干擾素樹突狀細胞活體內肌肉電擊法毒殺性T細胞活性
外文關鍵詞:Interferon betaDendritic cellin-vivo intramuscular shockCTL activity
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β型干擾素的基因治療已初步被證實具有抑制如神經膠質瘤與黑色素細胞瘤等腫瘤生長的能力, 除了促進抑癌因子p53及干擾素腫瘤細胞訊息傳遞之外, β型干擾素也被認為具有刺激專一性免疫力而達到抑制腫瘤生長的目的。樹突狀細胞是一種抗原呈現能力很強又能刺激引發專一性免疫反應的細胞,未成熟的樹突狀細胞具有強大的吞噬抗原能力,而經過刺激成熟的樹突狀細胞則有很強的抗原呈現能力, 並足以刺激體內產生專一性免疫進而消滅外來抗原。由於β型干擾素具有刺激樹突狀細胞大量表現CD80/86表面抗原, 進而促進第二級訊號刺激增強T細胞為主的細胞型免疫反應, 尤其引起毒殺性T淋巴細胞免疫反應。在本研究中我們利用β型干擾素除了本身能夠誘導腫瘤細胞的細胞凋亡達到抑制腫瘤生長外,並配合增強樹突狀細胞抗原呈現能力來促進專一免疫如毒殺T細胞的能力來達成抑制大腸直腸癌CT26的生長作用。
在本研究中,我們首先在活體外試驗中確認β型干擾素生物活性與次技術圖狀細胞大量表現CD80/86表面抗原, 在活體試驗中我們利用活體內肌肉電擊法將β型干擾素基因轉殖入具大腸直腸癌腫瘤之小鼠體內後測量基因表現的時間,並在基因表現的第三天亦即β型干擾素蛋白質在小鼠內表現最大量時以腫瘤皮下注射的方式將我們已具抗原專一性的樹突狀細胞免疫疫苗來進行免疫療法。結果顯示這種結合干擾素基因治療與免疫細胞疫苗的治療策略除了能有效的提升細胞激素表現與引起CTL 毒殺性免疫反應外, 在抑制腫瘤生長與延長小鼠生存時間都有明顯的效果。
由我們結果對照過去的文獻, 我們認為高轉殖效率的肌肉電擊法比起之前使用皮下注射基因轉殖能延長 β型干擾素的表現時間外, 利用轉殖β型干擾素基因治療來速進數突狀細胞免疫疫苗的策略不僅能增加專一性免疫反應外也較緊基因治療或僅有免疫疫苗治療更能有效的抑制腫瘤生長與延長小鼠生命, 對於未來進行完手術切除後的術後治療也頗具有潛力。
Interferon-β (IFN-β) gene therapy has been proved to suppress cell growth in many tumor cells such as glioma and melanoma. Not only it activate tumor suppressor gene such as p53, but also proved to enhance adaptive immunity against tumor cell growth. Dendritic cell (DCs) is an efficient antigen presenting cell (APC) to promote specific immune response. Immature DCs has a strong phagocytic activity, and is able to drive adaptive immunity against antigen if they are mature. Interferon-β stimulates DCs to maturation through high level expression of surface markers such as CD80/CD86. This enhances the delivery of secondary signals to T lymphocytes and promotes stronger T cell-specific cellular immunity, especially the cytotoxic T lymphocyte (CTL) immune response. In this study, we would like to study the synergistic effect of IFN-β gene therapy and DC vaccines to regress colorectal carcinoma. Besides, the timing for introduction of DC vaccine to cooperate with IFN-β gene expression for maximum killing was especially concerned.
The study initiated with a verification of interferon-β biological activity on the increased expression of the secondary signal CD80/CD86 on DCs and induction of apoptosis of colorectal tumor cell line, CT26, in vitro. Animal experiments were then carried out to investigate the synergistic effect of IFN-β and DCs with an animal model of BALB/c mice bearing CT26 tumor cells. IFN-β plasmids were intramuscular electroporated, and the peak expression of IFN-β protein was observed at day 3 after introduction. DCs, after uptaking of tumor debris, were administered subcutaneously (SC) on the day of IFN-β expression peak or at the same day as the introduction of IFN-β. Results indicated that the optimal condition for tumor regression was to introduce DCs on the day with the highest IFN-β expression. In addition, the combination of IFN-β gene and dendritic cell vaccination could enhance cytokine expression, IL-12, TNF-alpha, IL-6, and induce CTL immune response in BALB/c mice with colorectal carcinoma. The inhibitory effect on tumor growth and prolonged lifespan were observed after the treatmet. Intramuscular electroporation demonstrated higher transfection efficiency and extended gene expression than SC, which had been previously done at our lab. Moreover, the combination of DCs and IFN-β gene showed a much better suppression effect on tumor growth than DCs or IFN-β gene solely. Taken together, this combination may constitute a new therapeutic strategy to for anti-colorectal carcinoma.
目錄
圖目錄
中文摘要
英文摘要
第一章 緒論
壹. 前言
貳. 大腸直腸癌
參. 癌症的免疫療法
肆. 癌症的基因療法
伍. 癌症疫苗的新策略
陸. 樹突狀細胞
柒. β型干擾素
捌. 利用活體內電擊法轉殖基因
玖. 研究動機與治療策略
第二章 實驗材料
第一節. 重組IFN-β基因的構築
第二節. 基因在真核細胞中的表現與蛋白質活性
第三節. 免疫細胞的分離與培養
第四節. 動物實驗
第五節. 實驗用培養液與緩衝液的配置
第三章 實驗方法
第一節. IFN-β基因構築與選殖
第二節. 基因表現與蛋白質活性分析
第三節. 免疫細胞培養與表型分析
第四節. 動物實驗
第四章 實驗結果
第一節. IFN-β基因構築與選殖
第二節. 基因表現與蛋白質活性分析
第三節. 免疫細胞分離與分析
第四節. 動物實驗
第五章 討論
第六章 參考文獻
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