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研究生:蔡明憲
研究生(外文):Ming-ShianTsai
論文名稱:Oct4在肺癌中調控腫瘤相關巨噬細胞的角色
論文名稱(外文):Role of Oct4 in regulation of tumor-associated macrophages in lung cancer
指導教授:蕭璦莉
指導教授(外文):Ai-Li Shiau
學位類別:碩士
校院名稱:國立成功大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:42
中文關鍵詞:巨噬細胞腫瘤巨噬細胞刺激因子肺癌
外文關鍵詞:macrophagetumormacrophage colony-stimulating factorlung cancer
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M1/M2 腫瘤相關巨噬細胞 (tumor-associated macrophage, TAM) 的極化現象 (polarization) 與肺癌的進程以及預後之預測是有關係的。M1 巨噬細胞可以藉由毒殺機制以及分泌發炎性細胞激素抑制腫瘤生成,而M2巨噬細胞則是可藉由抑制免疫系統以及促進血管新生 (angiogenesis) 促進腫瘤生長。Oct4被認為是在調控幹細胞之分化多能性 (pluripotency) 中扮演著重要角色。近來的研究顯示,Oct4被證明大量表現在某些癌症之中,包括了肺癌和膀胱癌等,且在腫瘤進程扮演著重要的角色。Oct4可以藉由其轉錄活化 (transactivating) 的角色調控大量的基因,以及增進腫瘤的生長、抗藥能力和轉移 (metastasis)。然而,Oct4在免疫系統中所扮演的角色仍是未知的。在本研究中,我們探討Oct4在肺癌中調控腫瘤相關巨噬細胞的角色。我們使用了經過脂多醣體 (lipopolysaccharide, LPS) 刺激之THP1 血癌 (leukemia) 細胞做為M1巨噬細胞。首先,將M1 巨噬細胞與A549肺癌細胞進行共同培養 (co-culture) 可發現癌細胞中的Oct4有提高表現量的現象。接著以 M1所表現的促發炎細胞激素TNF-α和 IL-1β,刺激A549細胞也可發現癌症細胞內Oct4表現量上昇,這個現象可以經由加入NF-κB抑制劑JSH-23而消失。我們認為Oct4的上升可以去誘導一些抑制免疫系統的細胞激素,進而去抑制M1巨噬細胞的作用,甚至使M1轉變為可以促進腫瘤增生的M2巨噬細胞。我們發現了在Oct4大量表現的A549以及CL-1-5肺癌細胞中會調控M1巨噬細胞轉變為M2巨噬細胞的巨噬細胞刺激因子也大量上升 (macrophage colony-stimulating factor, M-CSF)。以Oct4大量表現的A549細胞培養液刺激M1巨噬細胞,也會提高巨噬細胞表現出M2的細胞標誌CD206。因此,我們認為Oct4在調控巨噬細胞的極化現象上,扮演著重要的角色,並且可以做為肺癌治療的潛力標誌。
M1/M2 tumor-associated macrophage (TAM) polarization is associated with lung cancer progression and prognostic prediction. M1 macrophages can inhibit tumor growth by exerting cytotoxic effects and secreting inflammatory cytokines, whereas M2 macrophages can promote tumor growth by inhibiting immune responses and promoting angiogenesis. Oct4 is considered as one of the key regulators of stem cell pluripotency. Oct4 has been demonstrated to be overexpressed in some human cancers, include lung cancer and bladder cancer. It plays an important role in cancer progression. Oct4 can regulate expression of a variety of genes by its transactivating role and promote tumor progression, anti-drug resistance and metastasis. However, the role of Oct4 in immune system remains unknown. In this study, we studied the role of Oct4 in regulation of TAM polarization in lung cancer. We used lipopolysaccharide (LPS)-treated THP1 leukemia cells to induce M1 macrophages. Coculture of M1 macrophages with A549 lung cancer cells resulted in the upregulation of Oct4 expression in cancer cells and secretion of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, in the conditioned medium. Furthermore, expression of Oct4 was upregulated in A549 cells treated with TNF-α and IL-1β, and such effects were abolished by treatment with the NF-κB inhibitor JSH-23. We hypothesized that upregulation of Oct4 may induce overexpression of immunosuppressive cytokines to inhibit the functions of M1 macrophages and even to render M1 macrophages skewing to M2 macrophages, thus promoting tumorgenesis. We found that macrophage colony-stimulating factor (M-CSF) was upregulated in Oct4-overexpressing A549 and CL1-5 lung cancer stable clones, and was capable of skewing M1 macrophages to M2 macrophages. Moreover, M1 macrophages expressed higher level of the M2 marker CD206 after treated condition medium of Oct4-overexpressing A549 cells. Taken together, Oct4 may have the critical role in TAM polarization and be a potential therapeutic target for lung cancer.
中文摘要 I
英文摘要 III
致謝 V
目錄 VI
圖目錄 VIII
縮寫 IX
第一章 緒論 1
1. 肺癌 1
2. Oct4 2
3. 免疫系統與腫瘤 4
第二章 研究目的 8
第三章 材料與方法 9
材料: 9
1. 質體 9
2. 細胞株 9
3. 寡去氧核甘酸 9
方法: 11
1. 細胞培養 11
2. 慢病毒之生產 12
3. 慢病毒之感染細胞 13
4. 西方墨點法 13
5. 免疫組織染色法 14
6. 反轉錄酶聚合連鎖反應 15
7. 細胞轉染實驗 16
8. 啟動子活性分析 16
9. Enzyme-link immmunosorbent assay 16
10. 轉形實驗 17
11. 流式細胞儀 17
12. 統計學 17
第四章 結果 19
一、M1巨噬細胞之誘導 19
二、M1巨噬細胞與A549肺癌細胞株共培養刺激A549內Oct4表現量上升 19
三、使用促發炎細胞激素刺激A549及LL/2細胞可使細胞Oct4表現量上昇 20
四、使用促發炎細胞激素刺激A549細胞可使Oct4啟動子活性增強 20
五、TNF-α及IL-1β 經由NF-κB 訊號傳遞路徑使 Oct4 表現量上升 21
六、Oct4可使A549及CL1-5分泌的M-CSF表現量提高 21
七、Oct4過量表現之A549細胞培養液可調控THP-1 M2巨噬細胞標誌 CD206之表現 21
第五章 討論 23
參考文獻 26
圖 31
附錄 41
自述 42
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