免疫治療為透過增強病患的免疫反應的一種療法，常見的方法有使用給予細胞激素、在體外活化可以攻擊腫瘤的免疫細胞從外部給予免疫能力的方式進行治療，另外也有給予免疫抑制劑、溶瘤病毒及利用病患自體樹突細胞作為遞送腫瘤抗原進而刺激病患的免疫能力。溶瘤腺病毒屬於溶瘤病毒的一種，特性為不具外套蛋白、攜帶35 kb 雙股DNA的一種病毒，臨床上已有利用溶瘤腺病毒進行治療，在實驗室先前所發表的結果中構築了以9倍Oct4 response element(9xORE)以及6倍缺氧誘導因子反應片段驅動的溶瘤腺病毒，命名為Ad.LCY，Ad.LCY不管在細胞實驗中能夠選擇性的毒殺腫瘤細胞，而不會攻擊正常細胞，並且以Ad.LCY治療帶有MBT-2膀胱癌腫瘤細胞的小鼠，比起未治療的組別有良好的抑制腫瘤生長的效果，且生存率方面也較高。樹突細胞免疫受體(Dendritic cell immunoreceptor,DCIR)是位在樹突細胞上的C型凝集素受體，會通過辨識外來或自身結構的碳水化合物片段，在免疫系統中表達功能，在先前的研究中，帶有MBT-2膀胱癌細胞以基因槍打入shCleac4a進而降低小鼠體內樹突細胞的DCIR表現量，結果中顯示以此進行治療，會有良好抑制腫瘤生長效果，並且也成功引起CD8+ T細胞的免疫能力。我們的研究致力於生產一個帶有免疫調節功能的溶瘤腺病毒，期望引起更強的免疫能力；首先我們構築Ad.LCY使其攜帶shClec4a基因片段，命名為Ad.shDCIR，接著使用慢病毒讓A549肺癌細胞表達DCIR並用Ad.shDCIR感染，確認Ad.shDCIR帶有降低DCIR表現量的功能；接著我們再以Ad.shDCIR直接感染小鼠體內的骨髓分化的樹突細胞，或是收集感染過Ad.shDCIR的MBT-2細胞培養基再培養樹突細胞，我們都可以觀察到相比未處理的組別DCIR的表現量都有下降的趨勢；接著以Ad.shDCIR感染MBT-2細胞以及NIH3T3小鼠的胚胎成纖維細胞，我們觀察到Ad.shDCIR只對癌細胞有毒殺效果，所以我們也確認了Ad.shDCIR是具有選擇性的。我們目前的研究不僅顯示Ad.shDCIR可以有效的毒殺MBT-2膀胱癌細胞，並且也能有效的降低DCIR的表現量；接著在動物實驗中觀察Ad.shDCIR與Ad.LCY皆有抑制腫瘤生長的效果，但二者抗腫瘤效果相似，後續可繼續評估抗腫瘤效果。

Bladder cancer is one of the most common malignant neoplasms in the genitourinary tract. Immunotherapy is often used as part of the standard treatment. It has been shown that tumor-
infiltrating CD8 + T cells are correlated with prognosis of bladder cancer. Oncolytic adenovirus has been exploited for cancer therapy due to its ability to selectively replicate in and lyse tumor cells. We have previously shown that oncolytic adenoviruses driven by 9 copies of Oct4 response element (ORE) combined with 6 copies of hypoxia response element (HRE) exhibit potent antitumor activity against bladder cancer cells, but spare normal cells. Dendritic cells can present captured antigens and induce immune responses. Dendritic cell immunoreceptor (DCIR) expressed in some immune cells, such as dendritic cells and macrophages, is an immune suppressor of dendritic cells. Previous studies have shown that silencing of DCIR enhances CD8 + T cell immunity and antitumor activity. In the present study, we generated a novel oncolytic adenovirus encoding short hairpin RNA (shRNA) targeting Clec4a, designated Ad.shDCIR, under the control of 9 copies of ORE combined with 6 copies of HRE aiming at inducing cytolysis of tumor cells and enhancing antitumor immunity. First, We constructed the oncolytic adenovirus Ad.shDCIR and demonstrated its effect on inhibiting DCIR expression in DCIR-overexpressing A549 human lung cancer cells. Secondly, we demonstrated that Ad.shDCIR also slectively killed murine MBT-2 bladder cancer cells, but not normal cells. Thirdly, we observed that expression of
DCIR was decreased in dendritic cells treated with the conditioned medium of MBT-2 murine bladder cancer cells infected by Ad.shDCIR. In the syngeneic MBT-2 tumor model, Ad.shDCIR could inhibit tumor growth in tumor-bearing mice. In conclusion, Ad.shDCIR may be further explored as an antitumor agent against various types of cancer.

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