臺灣博碩士論文加值系統

大腸直腸癌在全世界和臺灣均是癌症死亡原因的前三名。本實驗室先前的研究，於人類第四號染色體4q26鑑定NDST4於大腸直腸癌可能扮演抑癌基因的角色。NDST4屬NDST (N-deacetylase/N-sulfotransferase heparan glucosaminyl)家族的一員，主要的功能是參與Heparan sulfate (HS)的生合成。HS鍵結於蛋白多醣(proteoglycan, PG)的核心蛋白，即為heparan sulfate proteoglycans (HSPGs)。HSPGs廣泛分布於許多組織，主要存在於細胞膜、細胞內與胞外基質。HS能夠連結許多蛋白質配體(ligand)，包括生長因子、細胞激素和趨化素，進而調控許多生物活性。據此，實驗室製造Ndst4基因剔除小鼠，初步研究，此基因剔除鼠的發育和生育能力正常。本論文乃利用此小鼠模式探討Ndst4在急性大腸炎和發炎相關的大腸腫瘤生成所扮演的角色。我們將Ndst4異合子(N5F1)之公鼠與母鼠相互交配產生Ndst4基因剔除小鼠(N5F2)和野生型(wild-type,WT)小鼠。於急性大腸炎模式，我們選用六至八週WT及Ndst4基因剔除母鼠給予含3% dextran sodium sulfate (DSS)的飲用水連續七天，結果顯示Ndst4基因剔除小鼠體重下降程度、盲腸充血情形以及疾病活性指數(Desease activity index, DAI)皆較WT小鼠嚴重；進一步做組織切片染色，發現Ndst4基因剔除小鼠之遠端大腸黏膜層組織發炎程度比WT小鼠嚴重。另一方面，我們將四至六週WT及Ndst4基因剔除公鼠以腹腔注射12.5mg/kg azoxymethan (AOM)，接續給予含2.5% DSS的飲用水，以誘導發炎相關的大腸腫瘤生成，結果顯示：Ndst4基因剔除小鼠有明顯脫肛現象，於腫瘤生成的數量及直徑超過2mm的腫瘤數量明顯較多，且具有侵犯特性。進一步以免疫組織化學染色觀察正常隱窩(crypt)及腫瘤組織之細胞增生和凋亡情形， WT及Ndst4基因剔除小鼠無顯著差異。接著定量分析小鼠正常黏膜及腫瘤組織之細胞激素、趨化素及轉錄因子的表現量，結果顯示：Ndst4基因剔除小鼠之腫瘤組織TNF-α、CXCL1及COX2基因表現量均比WT小鼠低，Foxp3基因表現量則比WT小鼠高，然而IL-1β、IL-6、CCL2、iNOS、IFN-γ、IL-4、IL-17A、IL-10、T-bet、GATA3及Rorγt mRNA表現量無顯著差異。於正常黏膜組織則皆無明顯差異。綜合以上結果，缺乏Ndst4可能影響特定HSPG的修飾結構，導致形成適合腫瘤生長的微環境及促進腫瘤的侵襲性。

Colorectal cancer (CRC) is one of the most important causes of cancer death in the world and Taiwan. Our previous study has identified that NDST4 located at chromosome 4q26 is a putative tumor suppressor gene in CRC. NDST4 belongs to the N-deacetylase/N-sulfotransferase (heparan glucosaminyl ) (NDST) family, which are responsible for heparan sulfate (HS) biosynthesis on a core protein to form heparan sulfate proteoglycans (HSPGs). The HS chains of HSPGs bind to and regulate the functions of various growth factors, cytokines and chemokines. HSPGs ubiquitously reside on cell surface, inside the cell, and in the extracellular matrix. We further generated an Ndst4-knockout (Ndst4 KO) mouse strain, which could develop and reproduce normally. In the thesis, the role of Ndst4 in acute colitis and colitis-associated tumorigenesis was investigated via in the genetically engineered mice. Using acute colitis model induced by dextran sodium sulfate, Ndst4 deficiency resulted in a significantly body weight loss, higher disease activity index and obviously hyperemic appearance in the cecum when compared with wild-type (WT) mice. In addition, histological examination in distal colon also showed an increased level of crypt erosion in Ndst4 KO mice. In the colitis-associated tumor model induced by azoxymethane/dextran sodium sulfate, the number and size of colonic tumors were significantly increased as compared with WT mice. Furthermore, tumor invasiveness and the prolapse frequency were also significantly increased in Ndst4 KO mice. Meanwhile, cell proliferation and/or apoptosis in normal crypt and/or tumor burden were studied by using immunohistochemistry. However, there was no significant difference between WT and Ndst4 KO mice. On the other hand, tumor tissues exhibited significantly lower expression of TNF-α, CXCL1 and COX2 RNA transcripts and higher expression of Foxp3 RNA transcripts in Ndst4 KO mice by quantitative RT-PCR analysis, IL-1β, IL-6, CCL2, iNOS, IFN-γ, IL-4, IL-17A, IL-10, T-bet, GATA3 and Rorγt expression were not different between the two groups. The gene expression studied in normal parts was not different between the two groups. Taken together, loss of Ndst4 might impair the modification of HS chains of specific HSPGs leading to tumor-promoting microenviroment and tumor
invasiveness in the colon.

致謝 i
中文摘要 iii
英文摘要 v
縮寫對照表 vii
圖目錄 xii
1. 研究背景 1
1.1大腸直腸癌 1
1.2 抑癌基因 2
1.3 發炎性腸道疾病 2
1.4 發炎相關大腸癌 5
1.5 腫瘤免疫編輯 6
1.6實驗動物模式 8
1.6.1 Dextran sodium sulfate動物模式 8
1.6.2 Azoxymethane/Dextran sodium sulfate動物模式 9
1.7 NDST(N-deacetylase/N-sulfotransferase heparan glucosaminyl)家族NDST4 11
1.8 Heparan sulfate proteoglycan與發炎性大腸疾病 12
1.9 Heparan sulfate proteoglycan與癌症發生及轉移 13
1.10 實驗室先前研究結果 15
1.10.1 第四號染色體之失異型性檢測 15
1.10.2 Ndst4基因剔除小鼠 16
2. 研究目標 17
3. 材料與方法 18
3.1 實驗小鼠 18
3.2 DNA萃取 18
3.3 多重PCR (multiplex PCR) 18
3.4 給予DSS誘發產生急性大腸炎 19
3.5 給予AOM/DSS誘導產生發炎相關大腸腫瘤生成 19
3.6 糞便潛血檢測 19
3.7 急性大腸炎之評估 20
3.8 病理組織處理及染色 20
3.8.1 病理組織處理 20
3.8.2 蘇木紫-伊紅染色 20
3.8.3 免疫組織化學染色 21
3.9 RNA表現量分析 22
3.9.1 RNA萃取 22
3.9.2 反轉錄合成cDNA 22
3.9.3 定量反轉錄聚合&;#37238;連鎖反應 23
3.10 統計分析 24
3.11 試劑和抗體 24
4. 結果 25
4.1 利用DSS誘發急性大腸炎動物模式以探討Ndst4之角色 25
4.1.1 Ndst4基因剔除小鼠體重明顯下降且疾病活性指數較高 25
4.1.2 Ndst4基因剔除小鼠盲腸充血情況較嚴重且大腸總長度有較短趨勢 25
4.1.3 Ndst4基因剔除小鼠遠端大腸發炎程度較嚴重 26
4.2 利用AOM/DSS誘發發炎相關大腸腫瘤生成動物模式以探討Ndst4之角色 27
4.2.1 疾病分析 27
4.2.1.1 Ndst4基因剔除小鼠體重沒有明顯差異但脫肛情況較嚴重……...27
4.2.1.2 Ndst4基因剔除小鼠腫瘤生成數量較多且大於2mm的腫瘤所佔
的比例也較高……………………………………………………………..27
4.2.1.3 Ndst4基因剔除小鼠之惡性腫瘤佔的比例較高……………………28
4.2.1.4 Ndst4基因剔除小鼠之腫瘤細胞具侵犯特性………………………28
4.2.1.5 Ndst4基因剔除小鼠之正常隱窩及腫瘤細胞增生沒有顯著差異…29
4.2.1.6 Ndst4基因剔除小鼠之正常隱窩及腫瘤細胞凋亡沒有明顯差異…29
4.2.2 基因表現分析 30
4.2.2.1 Ndst4基因剔除小鼠腫瘤組織表現細胞激素TNF-α程度低於WT
小鼠………………………………………………………………………..30
4.2.2.2 Ndst4基因剔除小鼠腫瘤組織表現趨化素CXCL1程度低於WT小
鼠…………………………………………………………...……………....31
4.2.2.3 Ndst4基因剔除小鼠腫瘤組織表現酵素COX2程度低於WT小鼠31
4.2.2.4 Ndst4基因剔除小鼠之腫瘤組織表現淋巴球分泌之細胞激素IFN-
γ、IL-4、IL-17A及IL-10與WT小鼠無顯著差異……………....…..…32
4.2.2.5 Ndst4基因剔除小鼠之腫瘤組織表現轉錄因子Foxp3 mRNA 程度
高於WT小鼠………………………………………………………….….32
5. 討論 33
6. 圖 38
7. 參考資料 58

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