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標題: | 探討N-deacetylase/N-sulfotransferase 4於大腸直腸癌對未摺疊蛋白質反應和巨噬細胞分化之調節 Study of N-deacetylase/N-sulfotransferase 4-mediated modulation of unfolded protein response and macrophage differentiation in colorectal cancer |
作者: | Wan-Syuan Lin 林琬璇 |
指導教授: | 楊雅倩(Ya-Chien Yang) |
關鍵字: | 大腸直腸癌,NDST4,內質網壓力,未摺疊蛋白質反應,巨噬細胞分化, Colorectal cancer,NDST4,Endoplasmic reticulum stress,Unfolded protein response,macrophage differentiation, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 大腸直腸癌於臺灣已攀升為每年發生率最高之癌症。本實驗室先前的研究,於人類第四號染色體 4q26區域鑑定 N-deacetylase/N-sulfotransferase 4 (NDST4) 為大腸直腸癌相關之抑癌基因。目前已知NDST4參與Heparan sulfate proteoglycans (HSPGs) 生合成之過程,而HSPGs於人體之發育、生長、發炎反應、抵抗微生物侵襲、癌症發生等不同生理及病理機制皆扮演重要角色。本論文將探討NDST4於大腸直腸癌細胞對未摺疊蛋白質反應 (Unfolded protein response, UPR) 和巨噬細胞分化之調控。第一部份利用doxycycline誘導方式使大腸直腸癌細胞株HCT116表現NDST4,並檢測UPR相關基因表現量,結果顯示NDST4於HCT116細胞未明顯影響UPR相關基因之表現;進一步利用內質網壓力 (ER stress) 促進劑tunicamycin提高HCT116細胞之UPR相關分子表現,NDST4對於UPR相關分子之調控仍是無顯著變化。另外,檢測52對臨床大腸直腸癌檢體UPR相關基因表現,並依其NDST4表現量分為表現降低組以及正常組,結果亦發現UPR基因表現於此兩群組間無顯著差異,利用大腸直腸癌資料庫之分析亦支持NDST4與UPR相關分子之表現無顯著相關。第二部份關於巨噬細胞分化調控之研究,乃利用 THP-1細胞株以及人類周邊血液單核細胞建立巨噬細胞分化模型,藉由細胞激素刺激使其分化為M1或M2亞型,以細胞形態、M1和M2亞型特異性基因及表面抗原表現確認分化模型之建立,並作為實驗之陽性控制組,實驗組則以大腸直腸癌細胞之條件培養液取代細胞激素之誘導分化作用,結果顯示:表現NDST4之大腸直腸癌細胞的條件培養液可促進巨噬細胞分化成M1亞型。另外,利用免疫組織化學染色檢測azoxymethane/dextran sodium sulfate (AOM/DSS) 小鼠模型之大腸腫瘤組織切片,分析其巨噬細胞的數量和分型,結果顯示Ndst4基因缺失會減少腫瘤及其相鄰正常組織之巨噬細胞浸潤,特別是M1亞型顯著降低;然而於NDST4異體移植腫瘤模型則發現不論大腸直腸癌細胞是否表現NDST4,僅少數巨噬細胞浸潤於腫瘤部位。 Colorectal cancer (CRC) is the cancer with the highest annual incidence in Taiwan. In our previous study, we identified N-deacetylase/N-sulfotransferase 4 (NDST4) as a novel tumor suppressor gene located at chromosome 4q26. NDST4 is one of the pivotal enzymes responsible for heparan sulfate biosynthesis on a core protein to form heparan sulfate proteoglycans (HSPGs), which play important roles in development, inflammation and tumorigenesis. In the study, we further aimed to investigate NDST4-mediated modulation of unfolded protein response (UPR) and macrophage polarization in CRC. In the first part, using doxycycline to induce various levels of NDST4 expression in CRC HCT116 cells showed that NDST4 had no effect on expression of UPR-related genes. The consistant results were observed when tunicamycin, an ER stress inducer, was used to upregulate UPR-related gene expression. In addition, there were no significant correlations between the expression of NDST4 and UPR-related genes in 52 primary tumors collected and TCGA CRC datasets. In the second part for modulation of macrophage polarization, macrophage differentiation models were established by THP-1 and monocyte-derived macrophages (MDM) which obtained from human peripheral blood mononuclear cells, and determined by morphological change, subtype-specific gene expression and surface markers. The conditioned media harvested from NDST4- expressing HCT116 cells could promote the polarization of M0 cells toward M1 macrophages. In addition, previously established azoxymethane/dextran sodium sulfate (AOM/DSS) murine model of colitis-associated cancer were used to measure the macrophage phenotypes. The results revealed that Ndst4 deficiency significantly decreased macrophage infiltration and M1 polarization in both of tumors and adjacent normal tissues. Whereas, in a murine xenograft tumor model using HCT116 cells, only few macrophages were observed in tumor tissue sections. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/74545 |
DOI: | 10.6342/NTU201902717 |
全文授權: | 有償授權 |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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