利用小鼠模式探討Ndst4於DSS誘發急性大腸炎之角色

大腸直腸癌在全世界和臺灣均是癌症死亡原因的第三名，且在臺灣大腸直腸癌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鑑定NDST4於大腸直腸癌可能扮演抑癌基因的角色。NDST4屬於NDST (N-deacetylase/N-sulfotransferase)家族的一員，其功能主要是參與heparan sulfate (HS)的合成。HS支鏈會以蛋白多醣(proteoglycan)為核心蛋白鍵結其上，形成heparan sulfate proteoglycans (HSPGs)。HSPGs廣泛存在於許多組織，主要表現於細胞膜與細胞外基質。HS能夠連結許多蛋白質配體(ligand)影響訊息傳遞，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。根據文獻指出，Ndst4為參與HS修飾的酵素，而HSPG可藉由HS結合並調控多種細胞激素和趨化素作用，顯示Ndst4的功能可能間接調控腸道發炎反應進而影響腸炎相關大腸癌(colitis- associated cancer)之發生。因此，本論文利用dextran sodium sulfate誘發小鼠之急性大腸炎模式，以探討Ndst4於急性大腸炎發展可能扮演的角色。首先，我們選用六週至八週的野生型(wild type，WT)和Ndst4基因剔除母鼠給予含3% DSS的飲用水連續七天並觀察其疾病程度，結果顯示：相較於WT小鼠，Ndst4基因剔除小鼠體重下降程度較嚴重，且疾病活性指數(disease activity index)明顯較高。接著進行組織學檢查，發現：Ndst4基因剔除小鼠遠端大腸之組織學活性指數(histology activity index)較WT小鼠高。我們進一步定量分析小鼠大腸黏膜層之相關基因mRNA表現程度，結果顯示：Ndst4基因剔除小鼠之促發炎細胞激素Il-1β、Il-6、Tnf-α和趨化素Ccl2及Cxcl1的RNA表現程度高於WT小鼠；同時Ndst4基因剔除小鼠之T細胞相關細胞激素Ifn-γ、Il-17a、Il-22和Il-10，以及T細胞相關轉錄因子T-bet和Foxp3也有相同結果；然而Ndst4基因剔除小鼠之組織修復相關細胞激素Il-18 mRNA表現程度則低於WT小鼠。其他包括：Il-4、Il-21、Gata3和Rorγt之表現程度則與WT小鼠沒有差異。利用免疫組織化學染色分析大腸上皮細胞增生與凋亡情形，發現Ndst4基因剔除小鼠之遠端大腸上皮細胞增生程度低於WT小鼠，且遠端大腸上皮細胞凋亡程度高於WT小鼠。另外，WT小鼠與Ndst4基因剔除小鼠在誘發大腸炎後之存活分析則沒有明顯差異。綜合以上結果，Ndst4缺乏可能使特定HSPG結構修飾發生改變而影響相關發炎和組織修復調控因子的呈現與作用，進而增加個體促進大腸組織發炎的敏感性。

關鍵字

大腸直腸癌； Ndst4 ； Heparan sulfate ；急性大腸炎

並列摘要

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in the world and Taiwan. Nowadays, CRC is the cancer with highest annual incidence rate in Taiwan. In our previous study, we had identified NDST4 as a novel tumor suppressor gene located at chromosome 4q26 that is lost in CRC at a high frequency. NDST4 belongs to an enzyme family designated the N-deacetylase/N-sulfotransferase (heparan glucosaminyl), which are responsible for synthesis of heparan sulfate proteoglycan (HSPG) by participating in modification of heparan sulfate chains covalently attached to its core protein. HSPGs are ubiquitously expressed both on the cell surface and in the extracellular matrix, and their HS chains can regulate a wide variety of bioactive molecules, especially cytokines and chemokines. As a result, HSPGs have long been shown to participate in the pathological processes of colonic inflammation, which contributes to the development of colitis-associated cancer. Accordingly, we had generated an Ndst4 knockout (Ndst4-/-) mouse strain, which develops and reproduces normally. In the study, we aimed to explore the role of Ndst4 in acute colitis in mice. Six to eight week-old Ndst4-/- mice and wild-type (WT) mice were administered 3% dextran sodium sulfate in drinking water for seven days. We observed an increased weight loss in Ndst4-/- mice. For disease activity index, Ndst4-/- mice exhibited more severe symptoms than WT littermates. In addition, the distal colon of Ndst4-/- mice showed an increased level of histological activity index. Further, by quantitative RT-PCR analysis, we revealed that the distal colon tissues of Ndst4-/- mice displayed significantly higher expression of proinflammatory cytokines (Il-1β, Il-6, and Tnf-α) and chemokines (Ccl2 and Cxcl1) mRNA transcripts, coupled with elevated transcripts of T cell-associated cytokines (Ifn-γ, Il-17a, Il-22 and Il-10) as well as transcription factors (T-bet and Foxp3). Meanwhile, Ndst4-/- mice showed a lower expression level of Il-18 mRNA, a tissue repair-associated cytokine. Nevertheless, Il-4, Il-21, Gata3 and Rorγt expression were not different between the two groups. By immunohistochemical staining, Ndst4-/- mice exhibited decreased proliferation and enhanced apoptosis of colonic epithelial cells in distal colon after DSS-induced acute colitis. However, there was no difference in survival between WT and Ndst4-/- mice after DSS treatment. In conclusion, Ndst4 deficiency might give rise to defective modification of specific HSPGs and subsquenly alter the regulation of various ligands, resulting in the increase of susceptibility to colonic inflammation.

並列關鍵字

Colorectal cancer ； Ndst4 ； Heparan sulfate ； Acute colitis

參考文獻

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利用小鼠模式探討Ndst4於DSS誘發急性大腸炎之角色

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