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DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 楊雅倩 | |
dc.contributor.author | SHIOU-TING CHEN | en |
dc.contributor.author | 陳秀婷 | zh_TW |
dc.date.accessioned | 2021-06-17T03:35:59Z | - |
dc.date.available | 2028-02-12 | |
dc.date.copyright | 2018-02-22 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2018-02-12 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/69957 | - |
dc.description.abstract | 本實驗室先前利用失異合性 (loss of heterozygosity) 研究,於人類第四號染色體4q26區域篩選出N-deacetylase/N-sulfotransferase (NDST4) 為大腸直腸癌相關之抑癌基因。已知NDST4參與Heparan sulfate proteoglycans (HSPGs) 之生合成過程,而HSPGs在發育、生長和炎症反應、腫瘤發生等不同生理和病理機制皆扮演重要角色。本論文將探討NDST4對於大腸直腸癌腫瘤微環境之巨噬細胞分化和血管新生的調控。首先利用THP-1及U937細胞建立巨噬細胞分化之細胞模型,藉由M0、M1和M2亞型得的細胞形態、標的基因和細胞表面抗原確認其分化。接續以表現NDST4之大腸直腸癌細胞或其條件培養液與M0細胞共培養,結果顯示:表現NDST4之大腸直腸癌細胞及其條件培養液可促進巨噬細胞分化成M1亞型。此外,利用先前研究之異種移植腫瘤組織切片經CD68染色,結果顯示NDST4表現之大腸直腸癌細胞所形成之腫瘤,其腫瘤周圍巨噬細胞 (tumor-associated macrophage) 的數量較多。另一方面,以不同濃度doxycycline誘導大腸直腸癌細胞表現NDST4,確認NDST4表現會抑制細胞內和分泌至細胞外的促血管新生因子urokinase-type plasminogen activator (uPA) 的mRNA及蛋白表現。檢測我們收集的51對大腸直腸癌檢體,腫瘤的uPA表現量明顯高於其成對的正常黏膜組織 ; 利用不同來源之大腸直腸癌資料庫分析,顯示預後較差的病患其腫瘤uPA表現量明顯較高,NDST4表現量則明顯較低。最後,藉由人類細胞激素晶片分析,發現誘導表現NDST4之大腸直腸癌細胞的條件培養液,其Serpin E1、macrophage migration inhibitory factor (MIF)、IL-8、CXCL12及CXCL1含量下降。綜合以上,推測大腸直腸癌細胞表現NDST4可能藉由減少上述調節因子,進而影響腫瘤微環境之巨噬細胞分化及血管新生。 | zh_TW |
dc.description.abstract | In previous loss of heterozygosity (LOH) study, we explore N-deacetylase/N-sulfotransferase 4 (NDST4) as a novel tumor suppressor gene (TSG) at chromosome 4q25-q28.2 in colorectal cancer (CRC). 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. We have proposed that loss of NDST4 function might impair the biosynthesis of specific HSPGs leading to tumor-promoting inflammation and tumor progression. In the study, we further aimed to investigate NDST4-mediated modulation of macrophage polarization and angiogenesis in CRC. First, macrophage differentiation model were established with THP-1 and U937 cells. Macrophage subtype were determined by morphological change and surface marker expression measured by qRT-PCR and flow cytometry. CRC cells with inducible NDST4 expression, including a single stable clone (HCT116/NDST4-C5) and a mixed line (HCT116/NDST4-Mix), were established in previous study. CRC cells expressing NDST4 and the conditioned media harvested could promte M0 cells differentiation into M1-polarized macrophages. Meanwhile, previously established xenogrft tumor tissue sections were used to perform immunohistochemistry staining of CD68 (M0). The results revealed larger numbers of tumor-associated macrophage in NDST4 expressing xenograft tumor. In the second part, NDST4 expression in CRC cells decrease the gene expression and protein secretion of urokinase-type plasminogen activator (uPA). In addition, uPA upregulation was observed in 51 primary tumor collected when compared to their matched normal mucosa. By using 4 public CRC datasets, the uPA expression is signigicantly higher in the tumors of high risk group than that in the tumors of low risk group. In contrast, the NDST4 expression is significantly lower in the tumors of high risk group.
The conditioned medium from CRC cells expressing NDST4 exhibited a decreased level of Serpin E1、MIF、IL-8、CXCL12 and CXCL1 by cytokine array analysis. Taken together, NDST4 expression in CRC cells might decrease the synthesis and/or secretion of the mediators, and these modulate macrophage differentiation and tumor angiogenesis in tumor microenvironment. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T03:35:59Z (GMT). No. of bitstreams: 1 ntu-107-R04424012-1.pdf: 3592504 bytes, checksum: 9c7eac0be8f0f26ead7dac0db5f62a1b (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 致謝 i
摘要 iii Abstract iv 一. 研究背景 1 1. 大腸直腸癌 1 1.1 簡介 1 1.2 生成機制 2 1.2.1 染色體的不穩定性 (Chromosomal instability, CIN) 2 1.2.2 微衛星不穩定性 (Microsatellite instability, MSI) 2 1.2.3 CpG 島甲基化表現型 3 1.3 大腸直腸癌分子特徵與標靶藥物的綜合分類系統 3 1.4 癌症分期 5 1.4.1 Dukes 分期系統 6 1.4.2 TNM 分期系統 6 1.4.3 AJCC/UICC 分期系統 8 2. 抑癌基因 8 3. N-deacetylase/N-sulfotransferase 4 ( NDST4 ) 9 3.1 NDST家族與NDST4簡介 9 3.2 NDST家族相關研究 11 3.3 Heparan sulfate proteoglycan與癌症發生及轉移 12 4. 腫瘤免疫編輯 14 5. 腫瘤相關巨噬細胞(tumor-associated macrophages , TAMs) 16 5.1 TAMs簡介 16 5.2 TAMs相關訊息傳遞路徑及因子 17 5.3 TAMs與大腸直腸癌 18 6. 血管新生 (Angiogenesis) 19 6.1 血管新生簡介 19 6.2 腫瘤血管新生 (Neoangiogenesis) 20 6.3 尿激酶型血纖維蛋白溶解酶原活化因子 (urokinase-type plasminogen activator , uPA) 21 6.3.1 uPA簡介 22 6.3.2 uPA訊息傳遞機制 22 6.3.3 uPA與癌症 23 7. 實驗室先前研究結果 24 7.1 第四號染色體之失異合性檢測 24 7.2 NDST4相關之異種移植腫瘤的小鼠模式 25 7.3 Ndst4基因剔除小鼠 25 二. 研究目標 28 三. 材料與方法 29 1. 試劑、材料及抗體及細胞株 29 2. 細胞培養 30 3. 蛋白質的抽取及定量 30 4. 西方墨點法 30 5. RNA 萃取 31 6. 反轉錄合成互補 DNA 31 7. 即時定量聚合酶連鎖反應 32 8. NDST4條件培養液(Conditioned medium, CM)製備 33 9. 巨噬細胞分化 33 10. 流式細胞分析技術(Flow cytometry) 33 11. 免疫組織化學染色(Immunohistochemistry stain) 34 12. 人類細胞激素晶片檢測(Human Cytokine Array) 34 13. 臨床數據分析(Clinical database analyze) 35 14. 統計分析 35 四. 結果 36 1. 利用THP-1細胞建立巨噬細胞分化之細胞模型。 36 2. U937細胞建立巨噬細胞分化之細胞模型。 36 3. 表現NDST4之大腸直腸癌細胞及其條件培養液對於巨噬細胞分化的影響。 37 4. 探討表現NDST4異種移植腫瘤之巨噬細胞的分佈。 40 5. 探討表現NDST4對於CRC細胞分泌調節因子於條件培養液之影響。 41 6. 確認表現NDST4對於大腸直腸癌細胞生成促血管新生因子urokinase-type plasminogen activator (uPA)之影響。 42 7. 利用大腸直腸癌資料庫分析NDST4及uPA與病人預後之相關性。 43 五. 討論 44 六. 圖 49 七. 表 77 八. 參考文獻 79 九. 附錄 100 | |
dc.language.iso | zh-TW | |
dc.title | N-deacetylase/N-sulfotransferase 4表現於大腸直腸癌細胞對巨噬細胞分化和血管新生之調控 | zh_TW |
dc.title | N-deacetylase/N-sulfotransferase 4 expression modulates macrophage differentiation and angiogenesis in colorectal cancer | en |
dc.type | Thesis | |
dc.date.schoolyear | 106-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 莊雅惠,潘思樺,徐立中 | |
dc.subject.keyword | 大腸直腸癌,NDST4,巨噬細胞分化,血管新生,uPA, | zh_TW |
dc.subject.keyword | CRC,NDST4,macrophage differentiation,angiogenesis,uPA, | en |
dc.relation.page | 115 | |
dc.identifier.doi | 10.6342/NTU201800554 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2018-02-12 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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