摘要
Her-2/neu這個致癌基因在將近20%的乳癌病人中有放大以及過量表現的現象。也因為Her-2/neu在腫瘤上的專一性表現，因此Her-2/neu就被認為是值得用來做為免疫治療的標的物之一。在我們先前的研究中證實，我們成功地利用異種DNA疫苗結合不同的細胞激素來抵禦內生性表現neu的腫瘤細胞。到目前為止，我們仍在嘗試發展更強而有力的佐劑來增加neu DNA疫苗的免疫療效。
TSP-1(thrombospondin-1)是由五個胞外的結構域所組成的家族成員，並且參與了細胞與細胞之間和細胞與基質間的溝通。但也有報導指出，TSP-1在人類的樹突狀細胞(dendritic cells)是一個會自我調控的負調節因子，並且會去活化潛在的TGF-β。因為TSP-1在免疫功能上不尋常的角色，我們把TSP-1 siRNA與人類的N端neu接在一起，先利用西方墨漬法以及免疫細胞染色確定其neu有正常的表現以及TSP-1 siRNA的確有壓抑TSP-1的功效。我們也證明利用基因槍的確可以遞送TSP-1 siRNA到淋巴細胞中並且抑制TSP-1的表現。把這樣的一個DNA質體當作DNA疫苗去治療，我們發現的確可以增加免疫療效。我們的結果提供了一個新式的策略去增加neu DNA疫苗的療效。我們也分析打過疫苗的老鼠組別裡，脾臟細胞中細胞激素的變化，發現專一性表現在Treg (T regulatory cells)中一個重要的轉錄因子-foxp3，有明顯的下降。而且我們也發現在結合TSP-1 siRNA後，比較起單獨施打neu的組別，促使CD4+ T cells走向Th1細胞免疫(cellular immunity)的細胞激素IFN-γ也有所上升。為了更加屏除U6 promoter所帶來的免疫影響，我們亦利用TSP-1 scramble siRNA當作控制組別去更專一性地証實這個假說。結果也發現scramble siRNA可以reverse TSP-1 siRNA所造成的免疫現象。本篇研究證明了利用基因槍去破壞樹突狀細胞的TSP-1 的確可以增加DNA疫苗的療效。這讓我們提出結合TSP-1siRNA會增加免疫療效的原因是因為其降低了Treg以及增加Th1細胞的表現。

ABSTRACT
The HER-2/neu oncogene is amplified and overexpressed in 20% of primary human breast cancer and due to the tumor specificity of its overexpression. The HER-2/neu oncoprotein is considered one of the most attractive targets for immunotherapeutic intervention. In our previously study, we successfully used xenogeneic DNA vaccine combining variety cytokines against mouse tumor cells overexpressing endogenous p185neu. Until now, we still try to discover the more powerful adjuvant to enhance the therapeutic efficacy of neu DNA vaccine. Thrombospondin-1 (TSP-1) consist of a family of 5 extracellular domains that participate in cell-to-cell and cell-to-matrix communication and it was reported to be an autocrine negative regulator of human DCs and to activate in vivo latent TGF-β. Base on its unusual role in immune response, we constructed the plasmid expressing TSP-1 siRNA and human-cyto-N’-neu, then check the neu expression level and knockdown effect of TSP-1 siRNA by immunohistochemistry and western blotting. The results suggested that vaccinated mice with TSP-1 siRNA by gene gun could inhibit the TSP-1 expression in draining lymph node. We found that the human-cyto-N’-neu-TSP-1 siRNA has better therapeutic effect. Our results provide novel strategies to enhance neu DNA vaccine therapeutic efficacy. We also found that Treg specific marker, foxp3, decreased in splenocytes of human-cyto-N’-neu-TSP-1 siRNA vaccination group. The expression of CD4+ subset Th1 cytokine-interferon-γ, increased in the groups of mice combing with TSP-1 siRNA. In order to eliminate the immune response induced by U6 promoter, we fused human-cyto-N’-neu with TSP-1 scramble siRNA as a control group to confirm the hypothesis accurately. We proved that the TSP-1 scramble siRNA reversed therapeutic efficacy and immune response compared with TSP-1 siRNA. In this thesis, we found that the therapeutic efficacy was improved by delivering TSP-1 siRNA into dendritic cells with gene gun. The mechanism of immune response induced by human-cyto-N’-neu-TSP-1 siRNA may due to down-regulate of Tregs and up-regulate of Th1.

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