臺灣博碩士論文加值系統

大腸直腸癌為我國發生率第一名之癌症，主要是由於腸道上皮細胞不正常增生，並累積基因變異，包含抑癌基因失去功能和致癌基因過度活化所致。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。目前已知NDST4參與Heparan sulfate proteoglycan (HSPG) 生合成之過程，而HSPG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上皆扮演重要角色。實驗室先前建立以Tet-on inducible system表現NDST4之大腸直腸癌細胞株，研究結果顯示NDST4表現可以抑制異種移植腫瘤 (xenograft tumor) 之血管新生情形，接著利用human angiogenesis array篩選NDST4表現所調控之血管新生因子，結果發現urokinase-type plasminogen activator (uPA) 表現量在NDST4表現後顯著下降，因此本論文將探討NDST4對於大腸直腸癌血管新生情形之調控機制。以不同濃度doxycycline誘導不同NDST4表現量之大腸直腸癌細胞研究顯示：NDST4表現會藉由抑制NF-κB路徑之p65蛋白磷酸化而降低促血管新生因子uPA之基因及蛋白表現；且NDST4表現亦對於uPA參與之plasminogen activator (PA) 系統內其他成員造成相同趨勢之影響。另外，檢測其他血管新生相關因子之基因表現量，發現NDST4表現可以降低促血管新生因子angiogenin、amphiregulin、VEGFA及TIMP-1基因表現。以Phorbol myristate acetate 處理細胞增加NF-κB路徑活化以提高PA系統和促血管新生相關因子之表現後，發現NDST4表現對於uPA及其他因子有更明顯之抑制程度。

Colorectal cancer (CRC) is the top of cancer incidence in Taiwan, caused by abnormal proliferation of intestinal epithelial cells with accumulation of genetic alterations which include inactivation of tumor suppressor genes (TSGs) and activation of oncogenes. In previous loss of heterozygosity study, we identified N-deacetylase/N-sulfotransferase 4 (NDST4) gene as a novel CRC-associated TSG at 4q26. NDST4 plays a role in heparan sulfate (HS) biosynthesis, which modulates heparan sulfate proteoglycans (HSPGs) to regulate growth factors, cytokines and chemokines. Previously our lab has established Tet-On inducible NDST4-expressing human CRC HCT116 cells, and showed NDST4 expression exhibited significant inhibition of tumorigenesis with lower microvessel density in mouse xenograft tumor models. Accordingly, a Human Angiogenesis Array was used to identify the angiogenic factors regulated by NDST4 expression. The results revealed that urokinase-type plasminogen activator (uPA) was obviously decreased in the conditioned medium harvested from NDST4-expressing HCT116 cells. In the study, we further aimed to investigate the mechanism of NDST4 expression on regulating angiogenesis of CRC. Using different concentration of doxycycline to induce various levels of NDST4 expression in CRC cells showed that NDST4 could suppress gene and protein expression of pro-angiogenesis factor uPA via decrease of NF-κB p65 phosphorylation. Furthermore, NDST4 expression could show similar effect on plasminogen activator (PA) system, in which uPA is involved. On the other hand, NDST4 could also inhibit the gene expression of other angiogenesis-related factors, including angiogenein, amphiregulin, vascular endothelial growth factor A (VEGFA) and tissue inhibitor of metalloproteinase-1 (TIMP-1). After treating cells with phorbol myristate acetate, NF-κB pathway was activated, resulting in enhancement of the expression of PA system and angiogenesis-related factors in CRC cells. Then the inhibition effects of NDST4 expression on these genes were more obviously observed.

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
1.3 疾病分期 ......................................................................................................... 4
1.4 共識分子亞型Consensus molecular subtype (CMS) .................................... 5
1.5 治療方法 ......................................................................................................... 7
2. N-deacetylase/N-sulfotransferase 4 (NDST4) ...................................................... 9
2.1 NDST4與Heparan sulfate proteoglycan (HSPG)簡介................................... 9
2.2 NDST家族相關研究..................................................................................... 11
2.3 Heparan sulfate proteoglycan與癌症發生及轉移 ........................................ 12
3. 血管新生 (Angiogenesis) ................................................................................ 14
3.1 血管新生簡介 ............................................................................................... 14
3.2 腫瘤血管新生 (Neoangiogenesis) ............................................................... 14
3.3 尿激酶型血纖維蛋白溶解酶原活化因子 (urokinase-type plasminogen activator, uPA)...................................................................................................... 16
3.4 其他參與血管新生之因子 ........................................................................... 19
4. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) ......... 23
4.1 NF-κB簡介 .................................................................................................... 23
4.2 NF-κB訊息傳遞路徑 .................................................................................... 24
4.3 NF-κB與癌症 ................................................................................................ 24
5. 實驗室先前NDST4相關研究結果 ................................................................ 26
5.1 第四號染色體之失異合性檢測 ................................................................... 26
5.2 NDST4表現對腫瘤血管新生造成之影響................................................... 27
二. 研究目標 .............................................................................................................. 28
三. 材料與方法 .......................................................................................................... 29
1. 試劑、材料及抗體 ........................................................................................... 29
2. 細胞培養 ........................................................................................................... 29
3. 蛋白質抽取及定量 ........................................................................................... 30
4. 細胞增生 (cell proliferation) 分析 ................................................................. 30
5. NDST4條件培養液 (conditioned medium, CM) 製備 .................................. 31
6. 西方墨點法 ....................................................................................................... 31
7. RNA萃取 .......................................................................................................... 31

8. 反轉錄合成互補DNA ..................................................................................... 32
9. 即時定量聚合酶鏈鎖反應 ............................................................................... 32
10. TCGA大腸直腸癌公共資料庫臨床數據分析 .............................................. 33
11. 統計分析 ......................................................................................................... 33
四. 結果 ...................................................................................................................... 34
1. 排除NDST4 Tet-on inducible system所使用之doxycycline對於促血管新生因子 urokinase-type plasminogen activator (uPA)表現之影響。 .............. 34
2. 排除NDST4 Tet-on inducible system所使用之doxycycline對於 NF-κB路徑p65表現之影響。 ....................................................................................... 35
3. 決定誘導表現NDST4蛋白質之doxycycline濃度，並與正常組織之NDST4表現量相比較 ...................................................................................... 35
4. 確認表現NDST4之大腸癌細胞抑制uPA表現情形 .................................... 37
5. 探討表現NDST4之大腸癌細胞對NF-κB p65活化情形之影響 ................. 37
6. 利用PMA提高大腸癌細胞之uPA表現量後觀察NDST4表現對於uPA抑制現象 ............................................................................................................... 38
7. 探討以PMA處理大腸癌細胞前後NDST4表現之大腸癌細胞對於Plasminogen activator system其他成員之影響............................................... 40
8. 探討以PMA處理大腸癌細胞前後NDST4表現之大腸癌細胞對於其他促血管新生因子之影響 ....................................................................................... 41
五. 討論 ...................................................................................................................... 44
六. 圖 .......................................................................................................................... 51
七. 表 .......................................................................................................................... 80
八. 參考文獻 .............................................................................................................. 85
九. 附錄 ...................................................................................................................... 98

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