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系統識別號 U0026-0812200911120069
論文名稱(中文) 發展ErbB-2 DNA疫苗以改善基因療法對惡性 腫瘤之治療
論文名稱(英文) Immunogene Therapy of Tumors with DNA Vaccine Based on Autologous ErbB-2
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) of Biochemistry and Molecular Biology
學年度 92
學期 2
出版年 93
研究生(中文) 涂靜芬
研究生(英文) Cheng-Fen Tu
電子信箱 pattu@yahoo.com.tw
學號 s1691116
學位類別 碩士
語文別 中文
論文頁數 74頁
口試委員 口試委員-余俊強
口試委員-林以行
指導教授-賴明德
中文關鍵字 惡性腫瘤  疫苗 
英文關鍵字 vaccine  Erbb-2 
學科別分類
中文摘要   ErbB-2或稱Her2/neu 這個基因可以表現出一個185kD的穿膜醣蛋白,屬於上皮生長因子受體(epidermal growth factor receptor)家族中的一員,具有酪胺酸激酶(tyrosine kinase)的活性,在調控細胞生長、分化上扮演重要角色。在先前的研究中發現,許多人類癌症例如乳癌、卵巢癌、肺癌及膀胱癌中都可觀察到ErbB-2大量表現的情形,而此種蛋白質的大量表現和疾病的惡化以及難治癒率有很大的關聯性。由於ErbB-2為一腫瘤相關抗原(tumor-associated antigen)的特性,目前很多關於惡性腫瘤治療方式的研究就以ErbB-2為標的抗原(target antigen),希望藉由抑制ErbB-2這個腫瘤相關抗原的表現而達到抑制腫瘤生長的治療效果。其中DNA疫苗由於可以引發長期的免疫反應,目前為一發展中的可能的有效治療方式。然而,在先前的一些研究中指出,使用DNA疫苗這種主動免疫(active immunization)的方式時必須使用異種(xenogeneic) DNA才能克服小鼠體內的免疫耐受性(immunological tolerance),而引起免疫反應來對抗腫瘤細胞。本篇論文的目的是要探討,使用ErbB-2(Her2/neu)這種DNA疫苗時,是否必須使用異種(xenogeneic) DNA才能引發免疫反應,或是只要使用同種(autologous) DNA就能達到治療腫瘤的效果。

  為了探討使用N’-neu DNA疫苗是否必須使用xenogeneic DNA才能引發免疫反應而達到抑制腫瘤生長的治療效果,我們分別構築了N端extracellular domain的mouse p185neu和human p185neu這兩種質體DNA,命名為mN’-neu和hN’-neu。並以C3H/HeNCrj 品系的小鼠及其膀胱癌細胞株MBT-2作為研究模式。在我們的研究中顯示使用mouse N’-neu DNA疫苗這樣的同種(autologous) DNA疫苗作用在小鼠的腫瘤模式上,可以有效的抑制腫瘤的生長並延長小鼠的生存率。利用西方點漬的技術可以偵測到有施打DNA疫苗的小鼠血清中含有anti-p185neu的抗體。在腫瘤的切片中我們可以看到DNA疫苗可以增加一些免疫細胞如CD4+ T細胞、CD8+ T細胞在腫瘤浸潤的情形,也可以偵測到小鼠體內活化的CD8+ T細胞數目的增加。結果顯示mN’-neu與hN’-neu DNA疫苗的確可以引發小鼠體內的體液型免疫(humoral immunity)和細胞型免疫(cellular immunity)反應。在adoptive transfer的實驗中,我們觀察到從施打過DNA疫苗的小鼠取出的脾臟細胞的確可以抑制neu-positive腫瘤的生長,由此證實DNA疫苗所引發的細胞型免疫確實具有抗腫瘤的能力。在CD8+ T細胞去除後,DNA疫苗的治療效果就幾乎完全喪失了,因此,由此實驗更進一步說明DNA疫苗所引發的細胞型免疫在抑制腫瘤的治療效果上扮演相當重要的角色。

  在本篇研究中,我們證實mN’-neu 這種同種(autologous) DNA疫苗能夠引發小鼠體內的細胞型免疫(cellular immunity)反應,並且對於neu-positve的腫瘤有顯著的治療效果。對於有Her2/neu大量表現的腫瘤治療上,使用這種同種(autologous) DNA疫苗可能提供了一個新的可使用的策略。
英文摘要   ErbB-2 or Her-2/neu gene encodes an epidermal growth factor family receptor(EGFR) tyrosine kinase with oncogenic potential. The overexpression of ErbB-2 has been detected in many human tumors, including non-small cell lung cancer, breast cancer, and bladder cancer. Moreover, high levels of expression of this receptor have also been associated with tumor aggressiveness and poor prognosis. For these reasons, ErbB-2 is an attractive target for cancer immunotherapy, and a number of approaches have been investigated. Active immunization against ErbB-2 should be one of the useful approaches for the treatment of ErbB-2-positive tumor cells. However, the efficacy of DNA vaccine might be limited by the fact that ErbB-2 is a self Ag with poor immunogenicity due to immunological tolerance. Recently, it has been reported that the breaking of the immune tolerance against EGFR in a cross-reaction between the xenogeneic homologous and self EGFR. It remains to be investigated whether xenogeneic DNA is required for ErbB-2.

  To test the concept, we constructed the plasmid DNA encoding extracellular domain of xenogeneic (human) ErbB-2 (hN’-neu) and autologous (mouse) ErbB-2 (mN’-neu). The DNA vaccines were tested for the ability to induce antitumor immunity in ErbB-2-positive bladder cancer (MBT-2) in a mouse model (C3H/HeNCrj). Mice immunized with mN’-neu or hN’-neu showed therapeutic antitumor immunity. Sera isolated from the immunized mice recognized a single 185-kDa band in ErbB-2-positive tumor cells in western blot analysis. Massive increase infiltration of CD8+ T cells at the tumor site were observed in the mN’-neu and hN’-neu DNA vaccine groups of mice, while no infiltration of CD8+ T cells was observed in the control mice. To assess the quantity of neu-specific CD8+ T cells generated by vaccination with DNA vaccine, we performed intracellular cytokine staining with flow-cytometry analysis. Mice immunized with mN’-neu or hN’-neu generated higher frequency of neu-specific IFN--secreting CD8+ T cells than control mice. In addition, adoptive transfer of splenocytes showed the antitumor immunity. In vivo depletion of CD8+ T lymphocytes could almost completely abrogate the antitumor activity in the immunized mice, where as the depletion of CD4+ T lymphocytes showed partial abrogation. Our results indicated that the xenogeneic DNA vaccine might not be required for breaking immunological tolerance in a mouse animal model. The finding suggested that xenogeneic neu DNA is not required for potential human gene therapy in the future.
論文目次 緒論 1
一、致癌基因ErbB-2(Her2/neu)與惡性腫瘤之相關性 1
二、目前以ErbB-2(Her2/neu)圍標第抗原的免疫治療方式 2
三、使用DNA疫苗的優勢與限制 3
四、實驗目的與策略 5

材料與方法 8
一、細胞培養 8
二、質體製備 13
三、構築pRc/CMV-mN’-neu, pRc/CMV-hN’-neu 18
四、細胞免疫染色(Immunocytochemistry) 26
五、動物實驗 28
六、治療效果評估 29
七、去除CD4+, CD8+免疫細胞 41

結果 43
一、mN’-neu和hN’-neu質體DNA的構築及表現 43
二、mN’-neu和hN’-neu DNA疫苗對於腫瘤的治療效果 43
三、DNA疫苗所引發的體液型和細胞型免疫反應 44
四、探討CD4及CD8免疫淋巴球在疫苗治療效果的重要性 47

討論 48

結論 53

參考文獻 54

附圖與表 60
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