巨噬細胞是具有高度可塑性的免疫細胞，會因為其遇到的微環境而被極化為M1或M2巨噬細胞，對於維持生理功能的恆定性相當重要。Siglec-5和Siglec-14為主要表現於巨噬細胞上的配對型受體，兩者在配體結合位具有極高的相似性，能在相同配體的刺激下分別經由本身的細胞內ITIM motif (Siglec-5)或是結合具有ITAM motif的DAP12 (Siglec-14)來傳遞相反的訊息。此外，腫瘤細胞可藉由其唾液酸修飾蛋白去鍵結巨噬細胞上的Siglecs受體來調控巨噬細胞極化成腫瘤相關巨噬細胞的能力，因此，本篇論文主要想探討巨噬細胞上的Siglec-5/14配對型受體在巨噬細胞分別被極化為M1、M2或是腫瘤相關巨噬細胞時所扮演的角色。我們會先利用LPS/IFN-γ和IL-4/IL-13來建立巨噬細胞極化的模式，隨後會將巨噬細胞培養在腫瘤細胞培養液中來研究Siglec-5/14是否可以調控腫瘤相關巨噬細胞形成。首先，經由比較發炎性激素的產生，我們知道表現Siglec-5/14與否並不影響THP-1細胞在LPS/IFN-γ刺激下形成M1巨噬細胞的能力。但表現Siglec-14的THP-1細胞在IL-4/IL-13的刺激能夠表現比較多的M2指標，如CCL18、CCL22、IL-1RA、CD36和CD204，以及表現較高量可促使M2巨噬細胞極化的轉錄因子，如PPAR-γ和LXR-α。我們發現，實驗所用到的三種大腸癌細胞(SW480、SW620、HT29)培養液都能夠促進THP-1細胞形成腫瘤相關巨噬細胞，而肺癌細胞(CL1-0、CL1-5)和乳癌細胞(BT549)則無法極化THP-1成為腫瘤相關巨噬細胞。在大腸癌細胞培養液的處理下，與腫瘤相關巨噬細胞有關的指標會因THP-1細胞表現Siglec-5或是Siglec-14而有所不同。Siglec-14/THP-1細胞能夠產生出較多的CCL18、CCL22、IL-1RA、CD36、CD204和IDO，而Siglec-5/THP-1細胞會產生出較多的IL-10和MMP-9。經由分析多種轉錄因子的表現或是活性，我們認為Siglec-14可能可以在大腸癌細胞培養液的處理下經由促進STAT3的活化和LXR-α的表現來增加TAM指標的表達。總結來說，我們發現在巨噬細胞上的Siglec-5和Siglec-14受體的確能在刺激下影響巨噬細胞的極化，但目前仍不明瞭兩者會透過什麼樣的機制來進行調控。

Macrophages are highly plastic immune cells which can be further polarized into M1 or M2 macrophages in response to their surrounding microenvironment, and they play a critical role in maintaining the physiological homeostasis of the body. Siglec-5 and Siglec-14 are paired receptors primarily expresssing on macrophages and have the same sequence of amino acid in their first two N-terminal domains which are critical for ligand binding. However, Siglec-5 delivers inhibitory signals through its intracellular ITIM motif while Siglec-14 transduces activating signaling through the coupled ITAM-containing adaptor, DAP12, upon ligand engagement. In addition, sialylated glycoproteins derived from tumor cells have been shown to target various Siglec receptors to modulate macrophage polarizing into tumor-associated macrophages (TAMs) which can generate an immunosuppressive and pro-tumoral environment to assist cancer progression. In this study, we aim to investigate the role of Siglec-5 and Siglec-14 in macrophage polarization in response to different environmental cues. We first tried to establish a THP-1 macrophage polarization system driven by LPS/IFN-γ and IL-4/IL-13 stimulation, and a successful polarizing stimulation was proved by examing the expression of classical markers often used to classify the M1 and M2 population. Next, expression of M2/TAM markers of the THP-1 macrophages cultured with the tumor conditional medium were studied to explore the impact of the paired Siglec receptors, Siglec-5 and Siglec-14, in TAM programming. Comparable expression of M1 macrophage markers were observed in the Siglec-5/THP-1 and Siglec-14/THP-1 cells upon LPS/IFN-γ treatment, suggesting that exogenous expression of Siglec-5 and Siglec-14 on THP-1 cells did not affect M1 macrophage polarization. Notably, increased expression of M2-specific markers, CCL18, CCL22, IL-1RA, CD36 and CD204, as well as transcriptional factor involved in M2 polarization, PPAR-γ and LXR-α, were detected in Siglec-14-expressing THP-1 cells upon IL-4/IL-13 treatment. Moreover, we found that conditional mediums (CMs) derived from all the tested colorectal cancer cells (SW480, SW620 and HT29) successfully induce THP-1 macrophage differentiating into TAMs, while CM derived from lung cnacer cells (CL1-0 and CL1-5) and breast cancer cells (BT549) failed to achieve the same effects in our experimental condition. Importantly, Siglec-5 and Siglec-14 differentially regulated the TAM-related markers expression of the THP-1 macrophages stimulated with colon cancer CMs. Siglec-14/THP-1 cells expressed more CCL18, CCL22, IL-1RA, CD36, CD204 and IDO, while Siglec-5/THP-1 cells generated more IL-10 and MMP-9. Siglec-14 may induce the activation of STAT3 and the transcription of LXR-α to facilitate TAM formation upon colon cancer CM treatment. In conclusion, our results demonstrate that Siglec-5 and Siglec-14 can modulate the polarization of macrophages in response to IL-4/IL-13 and colon cancer CM, but further studies are required to elucidate the involved mechanisms.

口試委員會審定書 i
致謝 ii
中文摘要 iii
Abstract v
目錄 vii
第一章 研究背景與動機 1
一、巨噬細胞 (macrophages) 1
1. 巨噬細胞的功能 1
2. 巨噬細胞的極化 (macrophage polarization) 1
3. 巨噬細胞極化的分子機制 3
4. 巨噬細胞的極化與代謝作用 3
5. 腫瘤相關巨噬細胞 (tumor-associated macrophages) 4
二、Sialic acid-binding immunoglobulin-like lectins (Siglecs) 受體 5
1. Siglecs受體 5
2. Siglec-5和Siglec-14為配對型受體 (paired receptors) 7
3. Siglecs受體與巨噬細胞極化之間的關係 8
三、研究動機 9

第二章 研究材料與實驗方法 11
一、研究材料 11
1. 細胞株 (cell lines) 11
2. 重組蛋白 (recombinant proteins) 11
3. 酵素免疫分析法試劑組 (ELISA kits) 12
4. 抗體 (antibodies) 12
5. 引子 (primers) 13
二、實驗方法 14
1. 巨噬細胞的極化 14
2. 利用腫瘤細胞培養液處理巨噬細胞 16

第三章 研究結果 17
一、Siglec-5/14對於巨噬細胞極化的影響 17
1. Siglec-14/THP-1細胞在IL-4/IL-13刺激下表現較多M2巨噬細胞相關指標 17
2. IL-10和TGF-β1無法極化THP-1細胞形成M2巨噬細胞 18
3. IL-4/IL-13並不影響THP-1細胞唾液酸化之程度 18
4. Siglec-14/THP-1細胞在IL-4/IL-13刺激下表現較多的PPAR-γ和LXR-α 19
二、Siglec-5/14和腫瘤相關巨噬細胞之間的關係 20
1. Siglec-14受體可以促進巨噬細胞在大腸癌細胞培養液處理下TAM的形成 20
2. 多種癌細胞培養液對THP-1巨噬細胞形成TAM之探討 22
3. 大腸癌細胞培養液處理不改變TAM表面的唾液酸化程度 23
4. STAT3和LXR-α可能參與在Siglec-14下游來促進大腸癌細胞培養液處理所驅動的TAM極化 23

第四章 討論與未來研究方向 25
參考文獻 29
結果圖表 35

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