胰腺癌患者的五年存活率僅有5%，其免疫抑制的腫瘤微環境是難以治療的原因之一。胰腺癌細胞透過 PD-L1等免疫檢查點的表達，能逃避免疫細胞的辨識，免疫細胞因此無法攻擊癌細胞。本研究以體外實驗證實小分子藥物 DH-012能有效降低胰腺癌細胞膜上PD-L1蛋白之表達。另外，自小鼠脾臟或成人周邊血中分離出免疫細胞群，經過細胞激素誘導活化並放大，並使用細胞激素誘導殺手細胞毒殺胰臟腫瘤細胞。實驗結果發現，經過 DH-012處理後的胰臟腫瘤細胞對於細胞激素誘導殺手細胞毒殺較未處理組為敏感。透過檢測實驗組培養基中腫瘤壞死因子α 與干擾素γ，驗證腫瘤細胞的凋亡與免疫活化的程度呈現正相關。本研究為胰腺癌提供一項具開發潛力的治療策略。

The five-year survival rate of patients with pancreatic ductal adenocarcinoma (PDAC) is only 5%. The immunosuppressive tumor microenvironment is one of the reasons why it is difficult to treat PDAC. Pancreatic cancer cells can escape the recognition of T cells through the expression of immune checkpoint PD-L1. It leads to the disability of immune cells to attack cancer cells. In this study, we confirmed that small-molecule DH-012 could effectively decrease the expression profile of PD-L1 on pancreatic cancer cells. In addition, splenocytes or human peripheral blood mononuclear cell (PBMC) were activated by cytokine induction to produce cytokine-induced killer cells (CIK). It was found that under the treatment of DH-012, PDAC cells are more sensitive to the treatment of CIK than the untreated group. Through detecting the levels of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ), it was revealed that the degree of tumor cell apoptosis was positively correlated to immune response activation. This study can provide a potential therapeutic strategy for the treatment of PDAC.

610713011中文摘要 I
Abstract II
目錄 III
圖目錄 VII
表目錄 IX
一、 研究動機與研究目的 1
二、 研究背景介紹 3
2.1 胰臟癌 (Pancreatic cancer) 3
2.1.1 胰臟簡介 3
2.1.2 胰臟癌類型 3
2.1.3 胰臟癌發生率 4
2.1.4 診斷與治療 4
2.1.5 胰臟癌分期 5
2.1.6 整體存活率 5
2.1.7 致病原因 6
2.1.8 治療困境與腫瘤微環境 7
2.2 免疫療法 (Immunotherapy) 8
2.2.1 癌症免疫療法 8
2.2.2 免疫檢查點相關 9
2.2.2.1 免疫檢查點簡介 9
2.2.2.2 免疫檢查點PD-L1對胰臟癌臨床意義 9
2.2.3 細胞激素誘導殺手細胞 10
2.2.4 免疫檢查點抑制劑合併療法 11
2.2.5 小分子抑制劑 13
三、 研究材料與方法 15
3.1 實驗流程 15
3.2 癌細胞來源與培養 15
3.5 免疫細胞來源與培養 17
3.5.1 小鼠免疫細胞 17
3.5.2 人源免疫細胞 18
3.6 流式細胞分析 (Flow cytometry) 18
3.6.1 小鼠免疫細胞 18
3.6.2 人源免疫細胞 19
3.6.3 胰臟癌細胞 19
3.7 西方墨點法 (Western blot) 20
3.7.1 蛋白質萃取 20
3.7.2 蛋白質定量 20
3.7.3 膠體電泳、轉印與抗體呈色 21
3.8 反轉錄聚合酶連鎖反應 (RT-PCR) 21
3.9 細胞毒殺 (Cytotoxicity test) 22
3.10 酵素免疫分析法 (ELISA) 23
四、 研究结果與討論 25
4.1 胰臟癌細胞膜上 PD-L1表達受小分子藥物調控 25
4.1.1 胰臟癌細胞株 PD-L1表達 25
4.1.2 小分子藥物 DH-012降低胰臟癌細胞膜上 PD-L1蛋白之表達 26
4.1.3 小分子藥物 DH-012抑制 PD-L1基因表達 29
4.2 製備細胞激素誘導殺手細胞 (CIK) 31
4.2.1 鼠源細胞激素誘導殺手細胞 31
4.2.2 人源細胞激素誘導殺手細胞 35
4.3 細胞毒殺測試 40
4.3.1 存活率比較 40
4.3.2 分泌因子比較 45
4.4 機制初步探討 48
五、 結論 57
六、 參考文獻 59

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