臺灣博碩士論文加值系統

老鼠Cdx2基因，與果蠅caudal基因為同源性的基因，為含有homeodomain為其DNA-binding motif之轉錄因子。一些研究顯示在晚期的胚胎以及成體，Cdx2的表現侷限於前端大腸處之腸道上皮，同時Cdx2主導早期腸道的細胞增生、細胞分化以及細胞生長並保持腸道分化後的型態且與許多腸道癌症的形成有關，顯示了Cdx2對哺乳類腸道發育扮演著極端重要的角色。近年來斑馬魚為一新興的模式系統用來研究脊椎動物的胚胎發育過程，同時其與哺乳類可能具有相似的分子調節機制。因此本論文即以斑馬魚為研究材料來探討斑馬魚Cdx2同源基因是否亦在斑馬魚消化道的發育上扮演重要的角色。
利用斑馬魚基因庫- λgt10 cDNA library，得到可完整轉譯出255個胺基酸的斑馬魚Cdx2 cDNA，經由比對發現轉譯出的Cdx2蛋白質與西方有爪青蛙 (Silurana tropicalis) 的cad2有最高約69 % 氨基酸序列之相似度且包含靠近N-端的蛋白質活化區以及靠近C-端的保守區homeodomain。利用全覆式原位雜合反應來觀察Cdx2 mRNA在不同胚胎時期的表現，顯示Cdx2 mRNA首先出現於一細胞期，證實其為母體表現基因，表現持續至八細胞期、囊胚期、原腸胚期、尾芽體期至分節期，而原腸胚期時期起Cdx2 mRNA的胚胎表現主要集中於下胚層，且分節期在中後腦及部分體節有表現。受精後24小時，Cdx2 mRNA開始專一的表現於斑馬魚腸道之前端部分並逐漸向後端延伸，在48 hpf時表現於整條腸道。Cdx2 mRNA於腸道的表現在96 hpf之胚胎達到最高點，且其表現隨著胚胎發育至120 hpf及144 hpf有逐漸下降的趨勢。以48 hpf、72 hpf、96 hpf之斑馬魚冷凍切片來觀察此腸道專一性的表現，顯示其Cdx2 mRNA之表現主要集中於腸道頂端絨毛及基部細胞核等區域。
將反意寡核酸利用顯微注射入胚胎抑制Cdx2蛋白質的產生，在48 hpf時，注射過Cdx2專一性反意寡核酸的胚胎體成彎曲狀，且在120 hpf時可明顯發現軟骨性頭骨發育之不正常。進一步以全覆式原位雜合反應來觀察在注射過Cdx2專一性反意寡核酸的胚胎中各不同消化道標記基因表現的變異情形。如利用咽喉標記基因Shh及食道標記基因Dlx7來觀察兩者在咽喉與食道表現之變異情形，顯示了Cdx2基因功能之抑制會造成咽喉Shh mRNA之表現量及表現區有明顯的減少及不正常的現象且會造成Dlx7 mRNA在食道的表現量幾近於完全消失且使表現區有模糊不清且左右異位的現象；利用腸道標記基因Cdx2、IFABP及Gata6來觀察其在腸道表現的變異情形，顯示了Cdx2基因功能被抑制後會造成這些腸道標記基因的表現量大幅減少並使表現區域有不正常縮小且左右異位的現象；利用肝臟標記基因LFABP、HNF-4α、Gata6及Hhex來觀察其在肝臟表現的變異情形，顯示了Cdx2基因功能之抑制亦造成這些肝臟標記基因的表現量幾近消失並使表現區域沒有呈現正常新月型且左右異位的情形，而對另一個肝臟標記基因HNF-1α而言，HNF-1α mRNA在注射過胚胎中肝臟之表現沒有明顯的減少現象，但有左右異位的情形。利用外分泌性胰臟標記基因Trypsin及Gata6來觀察其在外分泌性胰臟表現的變異情形，同樣顯示了Cdx2基因功能的抑制會造成外分泌性胰臟標記基因的表現量減少並使表現侷限於一小區域及左右異位的情形；但利用內分泌性胰臟標記基因Insulin及Hhex來觀察其在內分泌性胰臟的表現，卻顯示了Cdx2基因功能的減少只造成內分泌胰臟的表現區域分散於兩處且有異位的情形產生但對其表現量是沒有影響。綜合上述，顯示Cdx2基因調控上述咽喉、食道、肝臟、外分泌性胰臟及腸道標記基因的表現並在這些消化器官發育左右不對稱性扮演著極端重要的角色。
為了了解Cdx2基因是否亦會調控其他左右不對稱發育的器官如:心臟的左右不對稱性分佈，進一步探討了表現於側板 (lateral plate) 中胚層左側之Lefty2基因以及表現於心臟的生長因子BMP4在被注射入Cdx2專一性反意寡核酸之胚胎中其在心臟表現的變異情形。所得結果亦顯示了Cdx2基因功能之抑制會造成心臟左右有異位的情形，綜上所知，Cdx2對斑馬魚器官左右不對稱之發育，扮演著上游調節性的重要角色。
Cdx2 基因亦為軟骨性頭骨正常發育所必需。以不同軟骨發育之標記基因來檢視注射過Cdx2專一性反意寡核酸的胚胎其軟骨性頭骨發育的過程。如利用檢視顱神經脊細胞遷移之標記基因Dlx2及Hoxa2來檢視顱神經脊細胞是否有正確的遷移情形，顯示Cdx2 基因功能的減少造成已遷移至菱腦原節鄰近與第二對及第三對咽弧軟骨之顱神經脊細胞中Dlx2及Hoxa2 mRNA的表現完全消失，證實了顱神經脊細胞沒有正確遷移的現象。利用Dlx7此標記基因來檢視顱神經脊細胞遷移至咽弧的過程，顯示Cdx2 基因功能之抑制對Dlx7 mRNA在咽弧的表現量並沒有影響，但其對第三至第七對咽弧表現區域之影響則顯示Cdx2 基因功能之喪失造成咽弧有融合在一起的現象；利用Chm1來檢視顱神經脊細胞遷移後分化成軟骨細胞之情形，結果亦顯示Chm1 mRNA在咽弧軟骨之表現量是不受影響的，證實了咽弧軟骨細胞之發育並沒有受到影響，但表現區域仍顯示Cdx2 基因功能之喪失造成咽弧有紊亂不正常的現象。另利用檢視軟骨細胞分化及提供軟骨發育所需膠原蛋白之標記基因Sox9a及Col2a1來檢視軟骨細胞是否有正常分化及型態形成的過程，顯示了Cdx2基因功能被抑制後其Sox9a及Col2a1 mRNA的表現量是正常的，證實了軟骨發育過程中軟骨細胞之分化現象及膠原蛋白的提供最後發育成為不同軟骨的過程是不受Cdx2基因功能喪失而有影響的，但其在軟骨性頭骨的表現區則顯示了Cdx2 基因功能之喪失造成咽弧軟骨不規則融合在一起且原來位於上方的髓顱軟骨和位於下方咽弧軟骨並沒有明顯的上下區隔而有髓顱軟骨與咽弧軟骨在下方重疊的現象。
綜合上述，顯示Cdx2 基因為髓顱及咽弧軟骨正常發育所必需，因為Cdx2 基因被抑制後會影響軟骨正常型態形成發育之過程並導致注射過的胚胎有咽弧軟骨不規則融合在一起且原來位於上方的髓顱軟骨和位於下方咽弧軟骨並沒有明顯的上下區隔而有髓顱軟骨與咽弧軟骨在下方重疊的現象。故Cdx2 基因之功能主要與顱神經脊細胞的遷移及遷移後於咽弧區域的定位有關，但對於顱神經脊遷移後咽弧軟骨細胞之形成、軟骨細胞的分化及軟骨基質的形成作用是沒有影響的。

目錄
 目錄………………………………………………..Ⅰ
 實驗方法目錄……………………………………..Ⅱ
 圖目錄……………………………………………..Ⅳ
 中文摘要…………………………………………...1
 英文摘要…………………………………………...4
 導論………………………………………………...8
 實驗方法…………………………………………..21
 結果………………………………………………..36
 討論………………………………………………..49
 參考文獻…………………………………………..54
 圖表………………………………………………..59
 附錄………………………………………………..88

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