C1GALT1調控的氧型醣化在SAS細胞中對單核球細胞分化的影響

Effects of C1GALT1-mediated O-glycosylation in SAS cells on monocyte differentiation.

杜思旻(Szu-Min Tu)

指導教授 ： 黃敏銓

共同指導教授 ： 林玫君(Mei-Chun Lin)

國立臺灣大學/醫學院/解剖學暨細胞生物學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201801801

摘要

頭頸癌在台灣十大癌症死亡率中排名第五，治療後復發轉移率高是造成頭頸癌死亡率高居不下的原因之一，頭頸癌患者整體五年存活率低於50％。異常醣化作用 (glycosylation) 會影響許多腫瘤細胞特性。Core 1 β1,3-galactosyltransferase (C1GALT1) 會調節氧型醣化作用 (O-glycosylation)，並促進頭頸癌、卵巢癌、乳癌的惡性特性。腫瘤細胞會分泌各種細胞激素至腫瘤微環境內，影響單核球細胞的移行與分化。已有研究顯示，腫瘤相關巨噬細胞會影響腫瘤細胞的生長、新血管生成和腫瘤微環境細胞外基質的重塑。此外，在臨床上也顯示腫瘤相關巨噬細胞與腫瘤的惡性程度和不良的預後相關。然而，頭頸癌細胞中的氧型醣基化對於單核球分化的影響目前還是未知的，因此，在本研究中我們探討在SAS頭頸癌細胞中由C1GALT1調控的氧型醣基化對單核球細胞分化的影響。首先我們利用免疫組織染色對41位頭頸癌患者的腫瘤切片進行分析，結果顯示當C1GALT1表現量較高時，在腫瘤附近的腫瘤相關巨噬細胞與第二型巨噬細胞數量皆較少。接著，我們利用流式細胞儀分析剔除C1GALT1後腫瘤細胞表面的醣型結構變化，證實剔除C1GALT1會影響腫瘤細胞的醣化作用，造成Tn antigen大量累積。為了模擬腫瘤微環境，我們使用SAS細胞的條件培養基 (Conditioned media) 分別刺激THP-1單核球細胞株與小鼠骨髓來源單核球細胞，分析單核球細胞的分化趨勢，結果顯示SAS細胞條件培養基會促進單核球細胞分化為第一型巨噬細胞，且抑制SAS細胞中C1GALT1的表現會加強此分化趨勢。另外，我們也證實了在SAS細胞中抑制C1GALT1的表現，會影響頭頸癌細胞中IL-6、CSF-1等細胞激素的mRNA表現量。最後我們構築了pET-29b/MPB-IL-6質體 (plasmid)，藉由大腸桿菌誘導產生MBP-IL-6重組蛋白，並使用質譜儀分析進行確認。未來，將會進一步製備IL-6多株抗體 (polyclonal antibody)，應用於IL-6的純化，期望可進一步研究不同醣型 (glycoform) 的IL-6在頭頸癌與免疫作用中扮演的角色。

關鍵字

頭頸癌 ； 醣化作用 ； C1GALT1 ； 單核球分化 ； IL-6

並列摘要

Head and neck cancer is the fifth of leading cause of cancer-related deaths in Taiwan. The majority of head and neck cancer treatment failures arise from metastasis and recurrence after surgical resection. The overall five-year survival rate remains less than 50%. Core 1 β1,3-galactosyltransferase (C1GALT1) controls the crucial step of mucin-type O-glycosylation. It has been reported to promote the malignant character of head and neck, bladder and breast cancer cells. Tumor cells secrete various cytokines into the tumor microenvironment (TME), recruit monocytes and regulate their differentiation to tumor-associated macrophages. In previous study, tumor-associated macrophages (TAMs) support diverse phenotypes within the primary tumor, including growth, angiogenesis and remodeling the extracellular matrix in TME. However, the effect of O-glycosylation in head and neck cancer cells on monocyte differentiation remains unknown. This study aims to determine the effects of C1GALT1-mediated O-glycosylation in SAS cells on monocyte differentiation. First, we analyzed forty-one sections of tumors from head and neck cancer patients by immunohistochemistry. The result showed that tumors expressing higher C1GALT1 contained more TAMs and M2 macrophages. Next, we showed that knockout of C1GALT1 changed the glycan structure on cell surfaces. To mimic the TME, we used conditioned media (CM) from SAS cells to induce differentiation of THP-1 and mouse bone marrow derived monocytes. Results showed that CM from SAS cells was able to skew monocytes into M1 macrophages, and knockdown of C1GALT1 enhanced this effect. Furthermore, knockdown of C1GALT1 downregulated IL-6 and CSF1 mRNA levels. In addition, the maltose-binding protein (MBP)-IL-6 recombinant protein was expressed in E.coli and we confirmed its identity by using mass spectrometry. In the future, polyclonal antibodies against IL-6 will be further prepared for the purification of IL-6. We hope to further study the role of different IL-6 glycoforms in the immunity of head and neck cancer.

並列關鍵字

head and neck cancer ； glycosylation ； C1GALT1 ； monocyte differentiation ； IL-6

參考文獻

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