2012-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/646872摘要：大腸直腸癌近年來在全世界及臺灣的十大癌症死因中皆排名第三。雖然目前的研究逐漸了解多步驟的基因變化在大腸直腸致癌過程中所扮演的角色，但對此癌症的篩檢與預防，以及病人治療與預後的評估仍是一個重要的挑戰。在我們先期的研究中，利用分布在第四號染色體上的22 個微衛星標記，針對106 例大腸直腸腫瘤進行失異合性(loss of heterozygosity, LOH) 分析，結果發現位於4q27 之D4S402 基因座的失異合性發生率最高，達32.9%。為了探究更精確的區域以尋找相關的抑癌基因，我們進一步以10 個微衛星標記建構4q25-4q28 區域（約12.9 Mb）的高解度刪除圖譜，定位出一個最小刪除區域，為1.33 Mb，最重要的是：此區域發生染色體漏失與大腸直腸癌的臨床分期有關，因此我們推測其存在有大腸直腸癌相關的抑癌基因。根據NCBI 資料庫，我們針對存在於這個區域中已知功能的基因進行初期研究，結果發現只有NDST4 基因在大腸直腸癌細胞株及腫瘤組織有表現量下降或完全不表現的情形，此結果顯示： NDST4很可能是存在於染色體4q26-4q28 中與大腸直腸癌相關的抑癌基因。此外，為了在動物模式中探討NDST4 基因的抑癌功能，我們也成功建立Ndst4 基因剔除小鼠，而此基因剔除鼠目前仍未在文獻中被發表過。藉由以上的研究進展，本計畫中，我們將鑑定NDST4 於大腸直腸之腫瘤生成與轉移之抑癌功能。本計畫研究目標如下:1. 確認NDST4 基因在大腸直腸癌表現受抑制，及了解其基因抑制的機轉；並分析NDST4 基因變化與大腸直腸癌臨床特徵及預後的相關性。2. 利用表現NDST4 cDNA 載體轉染不表現此基因的大腸直腸癌細胞，建立腫瘤抑制基因重新表現之細胞模式，並進行癌細胞的生物特性研究，以確定其抑癌基因的活性。3. 獲得以C57Bl/6 為遺傳背景之Ndst4 剔除鼠的同源近親品系(congenic strain)，並進行Ndst4 剔除鼠的表現型鑑定。4. 建立移植腫瘤到嚴重聯合免疫缺陷 (SCID) 小鼠的動物模式，以進一步驗證NDST4的腫瘤抑制功能。5. 於Ndst4 剔除鼠及Ndst4-null Apcmin/+小鼠模式，以致癌藥物dimethylhydrazine 誘發大腸直腸腫瘤，探討Ndst4 之抑癌功能。<br> Abstract: Colorectal cancer (CRC) is the third leading cause of cancer-related mortality in theworld and Taiwan. Colorectal carcinomas are characterized by the emergence ofmultiple chromosomal aberrations. By allelotyping with 22 polymorphic microsatellitemarkers spanning from chromosome 4pter to 4qter, we had defined the most frequentloss of heterozygosity locus D4S402 (4q27), which occurred allelic imbalance in morethan 32% of 106 colorectal tumors. Fine deletion mapping of 4q25-4q28 in thesecarcinomas further identified a minimal region of putative tumor suppressor loci withthe estimated genetic length of 1.33Mb at 4q26. Most importantly, deletion of thisregion is associated with late stage progression of CRC. Based on the results, wehypothesize that there are CRC-associated tumor suppressor gene(s) (TSGs) atchromosome 4q26. By searching with the NCBI database, there are three genes locatedin the minimal deletion region. By preliminary screening with RT-PCR andquantitative RT-PCR, NDST4 is the only one that gene expression is detected innormal colon mucosa, but is down-regulated in CRC cell lines and primary tumors. Inaddition, immunohistochemistry staining of CRC tissues also shows the similar result.For investigating the tumor suppressor function in vivo, we have also generated anNdst4 knockout (KO) mouse strain that has not yet been reported in literature so far.With these progresses, in this project, we pursue to identify NDST4 as a TSGinvolving in colorectal tumorigenesis and metastasis. Five specific aims are proposedas follows:Aim 1: To verify the gene inactivation of NDST4 in CRC tissues and to assess theclinical relevance of NDST4 gene alterations.Aim 2: T o ascertain tumor suppressor activities of NDST4 with in vitro cell model.Aim 3: To create a congenic strain of Ndst4 KO mice onto C57Bl/6 background and tocharacterize the phenotypes of Ndst4 KO mice.Aim 4: To ascertain tumor suppressor activities of NDST4 with in vivo xenografttumor model in SCID mice.Aim 5: To explore the role of NDST4 in dimethylhydrazine-induced colontumorigenesis in genetic mouse models, Ndst4 KO and Ndst4-null Apcmin/+mice.