摘要

　　酪胺酸激酶受體( receptor tyrosine kinase )對於對抗腫瘤的生成是一個重要的治療標的( target )。而c-Met這跟致癌基因和Her2/neu一樣是屬於其中的一員，媒介著肝細胞生長因子( hepatocyte growth factor )的致癌活性。有別於Her2/neu在體內的低表現，體內許多器官都可以發現到c-Met的表現，主要是調控著上皮細胞和內皮細胞的生長、分化、細胞移動及型態上改變。在先前的研究中發現到許多人類的癌症，例如乳癌、胃癌及肝癌的病患中，都有c-Met大量表現的情形，而這種蛋白質大量表現的情況和腫瘤的惡化、侵略性的生長與癌細胞轉移有著很大的關連性。而目前針對c-Met來設計的治療方式包括: 肝細胞生長因子之拮抗藥NK4、誘餌Met( decoy Met )以及其他抑制劑來阻斷這條訊息傳遞路徑而達到抑制腫瘤生長的效果。另外在先前研究發現，利用Her2/neu為標的抗原( target antigen )所設計的DNA疫苗能夠引發長期的免疫反應，而達到抑制腫瘤生長的治療效果。而c-Met和Her2/neu同屬於腫瘤相關性抗原( tumor-associated antigen )，本篇論文的第一個目的則是要先探討由c-Met所製備的DNA疫苗能否引發免疫反應來對抗腫瘤生長。另外，在先前研究也指出，使用DNA疫苗這種主動免疫( active immunization )的方式必須是異種( xenogeneic ) DNA才能克服小鼠體內的免疫耐受性( immunological tolerance )才能引起免疫反應。所以另一個目的則是利用c-Met DNA疫苗來探討是否必須利用異種DNA疫苗才能引發免疫反應。

　　為了探討c-Met DNA疫苗能否引起免疫反應以及是否必須是異種DNA疫苗才能產生療效，我們分別構築C端intracellular domain 的mouse Met和human Met這兩種質體DNA，分別能夠表現Met蛋白質的細胞內部分，命名為m-C’-met及h-C’-met。並以C3H/HeNcrj品系小鼠及其膀胱癌細胞株MBT-2和C57BL/6品系小鼠及其黑色素瘤細胞株B16作為研究模式。分別利用肌肉注射及基因槍兩種DNA傳遞方式送到小鼠體內，來評估其免疫反應及治療效果。從我們的研究顯示在肌肉注射方面，不管使用同種mouse-C’-Met或是異種human-C’-Met都能在MBT-2模式下產生療效，而在B16模式下則無法產生效果。然而在B16模式下，去除CD25+ T細胞後，可以發現到兩種Met DNA疫苗都可以引起免疫反應而產生療效。相對地，在基因槍傳送方式下，不論是在MBT-2或是B16模式下都能發現到異種h-C’-met DNA疫苗( xenogeneic )和同種m-C’-met DNA疫苗( autologous )都具有療效，而異種的DNA疫苗對於腫瘤似乎具有更好的效果，能夠有效抑制腫瘤生長並延長小鼠的生存。在腫瘤切片中我們可以看到met DNA疫苗都能夠增加一些免疫細胞例如CD4+ T細胞、CD8+ T細胞及NK細胞在腫瘤上浸潤的情形。

　　在本篇研究中，有別於neu基因，我們發現利用在體內普遍表現的c-Met基因來製備的DNA疫苗，不管是同種或是異種也都可以在某些條件下引發免疫反應，並對於met-positive的腫瘤有治療效果。

Abstract

　C-met proto-oncogene is a receptor tyrosine kinase that mediates the oncogenic activities of the hepatocyte growth factor (HGF) which is a pleiotropic growth factor involved in a variety of cellular responses, such as proliferation, migration, survival, and morphogenesis of epithelial, endothelial, and neuronal cells. C-met is also expressed in the epithelial cell of many organs, and overexpression of c-met has been correlated with tumor progression, metastasis and poor prognosis in breast cancer, gastric cancer, hepatocellular cancer, and melanoma. For this reason, c-met is identified as a broadly expressed tumor-associated antigen and an attractive target for cancer therapy. A number of approaches have been investigated to block this pathway to inhibit tumor growth. The first aim of this thesis is to investigate whether c-met DNA vaccine can induce antitumor immunity. Recently, it has been reported that only xenogeneic DNA vaccine can break immune tolerance to induce immune response. The second aim of this thesis is to study whether xenogeneic DNA vaccine is required for c-met. To test the concept, we constructed the plasmid DNA encoding cytosolic domain of human met (h-C’-met) and mouse met (m-C’-met). The DNA vaccines were tested for the ability to induce antitumor immunity in met-positive melanoma (B16) in a C57BL/6 model and bladder cancer (MBT-2) in C3H/HeNcrj model by intramuscular injection and gene gun bombardment. The results indicated that both m-C’met and h-C’-met could induce therapeutic effects in MBT-2 model by intramuscular injection, while the infiltrations of CD4+, CD8+ T cells and NK cells were observed in the tumors. In contrast, met DNA vaccines did not have therapeutic effects in B16 model by intramuscular injection. However, by elimination of CD25+ T cells to break immune tolerance, met DNA vaccines could also induce antitumor immunity in B16 model. In contrast, m-C’-met and h-C’-met had therapeutic effects by gene gun both in two models, and h-C’-met showed better therapeutic effects, while the infiltrations of CD4+ and CD8+ T cells were also observed in the tumors. The finding indicated that broadly-expressed c-met can serve as a target for DNA vaccine to induce therapeutic effects and xenogeneic form of DNA is not required for c-met DNA vaccine.

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