在生物體內，棘狀細胞是最主要的抗原呈現細胞，且具有活化專一性抗癌免疫反應之能力。將基因疫苗與以棘狀細胞為基礎之細胞疫苗合併應用已成為有效的癌症免疫治療策略。似Fms酪氨酸活化酶-3 配合體(Flt3L)與顆粒細胞¬-巨噬細胞叢聚刺激因子(GM-CSF)曾經被應用來促使棘狀細胞增生。過去的研究指出，結合載有GM-CSF及乳腺癌細胞抗原HER2/neu之質體(pN-neu)作為DNA疫苗，可引起由CD4胸腺細胞(T cells)主導之抗腫瘤免疫反應。在本篇研究當中，我們藉由可表現Flt3L及GM-CSF兩種細胞激素之質體(pFLAG)及合併兩個僅能單獨表現其中一種細胞激素之質體(pFL plus pGM)作為疫苗之基因佐劑來研究佐劑修飾作用於疫苗引起的免疫反應。我們更進一步嘗試將pFLAG經由真皮層注射來免疫小鼠，並希望外送之基因產物可以吸引蘭氏細胞及真皮層棘狀細胞浸潤到注射疫苗處，捕捉外送之pN-neu所表現出之特殊抗原。直接將DNA以真皮層注射方式可以將基因傳送進皮膚細胞，但過去的研究報告指出，利用真皮層注射一般實驗中所採用之七至八週齡小鼠，與同樣方式處理的四到五週齡小鼠相比，其基因傳送效率是大為降低的。因此我們也研究DNA疫苗配合高離子濃度溶液，是否可以加強基因表現並引起較佳之免疫反應。我們更近一步來研究單一質體pFLAG及合併兩個質體pFL和pGM其增強HER2/neu DNA疫苗(pN-neu)產生之免疫反應效用。在我們的實驗中發現，共同表現Flt3L及GM-CSF明顯有助於脾臟中棘狀細胞的成熟度及呈現抗原之能力。小鼠接受肌肉注射疫苗後(pN-neu及pFLAG)，其注射處引起棘狀細胞浸潤，增強小鼠之脾臟細胞針對腫瘤細胞分泌高量干擾素-丙(interferon-γ)，而細胞毒殺能力也明顯提升。當小鼠經由肌肉或真皮層注射pN-neu和pFLAG後，其體內產生了由CD8+ T細胞主導之針對自然表現出HER2/neu之小鼠膀胱癌細胞的抗腫瘤免疫反應。整體來說，我們的研究發現利用共同表現Flt3L及GM-CSF之策略具有轉移免疫反應類型之能力，且由pFLAG作為HER2/neu疫苗佐劑其產生之保護效果也優於合併使用兩個分開攜帶Flt3L和GM-CSF之質體。另一方面來說，我們也推測給予小鼠經由真皮層注射溶於高離子濃度溶液之疫苗後，體內所產生之抗腫瘤免疫反應與溶液協助轉殖基因快速且較長期的表現是具有相關性的，可以對於抗腫瘤疫苗的應用上有所助益。

Dendritic cells (DC) are powerful antigen-presenting cells for activation of specific antitumor immune responses. DNA vaccine and DC-based vaccine have emerged as promising strategies for cancer immunotherapy. Fms-like tyrosine kinase 3-ligand (Flt3L) and granulocyte-macrophage colony stimulating factor (GM-CSF) have been exploited for the expansion of DC. It was reported previously that combination of plasmid encoding GM-CSF with HER2/neu DNA vaccine induced predominantly CD4+ T cell-mediated antitumor immune response. In this study, we investigated the modulation of immune responses by murine Flt3L and GM-CSF, which acted as genetic adjuvants in the forms of bicistronic (pFLAG) and monocistronic (pFL and pGM) plasmids for HER2/neu DNA vaccine (pN-neu). Furthermore, we attempted to immunize mice with pFLAG intradermally to attract Langerhans cells and dermal DC infiltrating into injection site to capture the pN-neu encoded gene product. Intradermal injection of naked DNA results in gene transfer to skin cells, but previous report showed transfection efficiency in older mice (approximately 7 weeks) was a significant decrease in gene expression compared with younger mice (4-5 weeks old). We study whether DNA vaccine diluted in modified solution will enhance transgene expression and affect immune response compared with those diluted in traditional isotonic buffer. We further investigated the ability of pFLAG and pFL plus pGM for enhancing the efficacy of HER2/neu DNA vaccine (pN-neu). We found coexpression of Flt3L and GM-CSF significantly enhanced maturation and antigen presentation abilities of splenic DC. Increased numbers of infiltrating DC at the immunization site, higher interferon-γ production, and enhanced cytolytic activities by splenocytes were prominent in mice vaccinated intramuscularly with pN-neu in conjunction with pFLAG. Importantly, a potent CD8+ T cell-mediated antitumor immunity against bladder tumors naturally overexpressing HER2/neu was induced in the mice received intramuscular and intradermal vaccination with pN-neu and pFLAG. Collectively, our results indicate that bicistronic plasmid pFLAG modulate the class of immune responses and may be superior to those codelivered with pFL plus pGM as the genetic adjuvants for HER2/neu DNA vaccine. In other, we suggest vaccine-high ionic solution promote strong antitumor immune response in vaccinated mice that is associated with the rapid and prolonged expression of transgene may be a benefit in application of antitumor vaccine.

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