臺灣博碩士論文加值系統

研究目的:實驗室先前發表的研究顯示，Daxx過量表現時，會抑制腫瘤細胞生長。此研究嘗試尋找與Daxx蛋白相關的基因以及是否與大腸直腸癌發展的關聯性。
研究材料與方法: 藉由Daxx knock down的穩定細胞株以及微陣列篩選與即時定量PCR的確認，篩選出在大腸直腸癌細胞中可能受到Daxx表現調控的基因。進一步分析139組經由外科手術切除大腸直腸癌的病患配對檢體，利用西方點墨法，偵測蛋白表現量並搭配與臨床病例資料進行比對分析。
研究結果: 結果顯示顯示Daxx蛋白在大腸直腸癌檢體組織細胞中相較於正常細胞有明顯的表現減少。而篩選出的EHF蛋白的表現在在Daxx knock down的大腸直腸癌細胞株當中同樣出現明顯表現減少。分析EHF蛋白在大腸直腸癌組織檢體中整體表現為在腫瘤處表現相較於正常組織而言表現較低。EHF蛋白表現較高組共有34位(佔33.7%)而EHF蛋白表現量較低共有67位(佔66.6%)進一步針對臨床參數以及EHF
白的表現進行分析。結果顯示，在侵犯深度方面，在T1期的組織檢體中EHF表現比值為0.87；在T3期的組織檢體中，EHF蛋白的表現比值為0.59；而在T4期的組織檢體中EHF表現比值為0.46。就整體結果而言，侵犯深度較深的期別組織檢體中EHF蛋白普遍表現較低，且具有統計上顯著性（P＜0.05）。
結論:在人類大腸直腸癌細胞中，Daxx蛋白的表現減少時，EHF蛋白的表現會出現明顯的減少。而EHF蛋白的表現減少與臨床腫瘤侵犯深度有顯著的相關性。由此結果可知EHF蛋白可能受到Daxx蛋白的調控並且在大腸直腸癌當中與腫瘤侵襲深度有重要的相關性。詳細的調控路徑仍有待進一步的研究來了解。

Objective：
Colorectal cancer is one of the most lethal malignancy worldwide. Wnt signaling pathway dysregulation is observed in a majority of oncogenicity colon cancers. We previoud report Overexpression of death-associated protein altered the expression of Wnt downstream genes of Tcf4, including cyclin D1. However, themolecular mechanism underlying the biological function of Daxx in colorectal cancer is still unclear.

Methods and Materials：
By using established Daxx knock down cell line model and microarray, we found specific Gene regulated by Daxx. We further investigated DAXX expression in CRC cell line and in 101 matched clinical surgery pathologic sample pairs of CRC and adjacent normal tissue by Western blotting.
Results：
The results revealed that DAXX expression was significantly lower in the patients with CRC. Also we found when CEA is higher than 5ng/ml, Daxx protein is more likely to have lower expression in tumor site. Through th
e Daxx knock down cell line model , we found EHF gene is likely to be reg
ulated by Daxx protein. The decreased expression of EHF was found in
CRC patient sample( EHF expression ratio, mean:0.97, median:0.69) and
is associated with higher depth of invation(p<0.05)
Conclusion and Suggestion：
We found that ETS homologous factor (EHF), a member of E26 transform
ation specific (ETS) transcription factors EHF may be regulated by Daxx pr
otein in colorectal cancer tissues compared with non-tumorous tissues. T
ogether, both protein may have major effect of of oncogenicity colon ca
ncers. Moreover, decreased EHF expression level was correlated with mo
re severe tumor invation stage of patients with colorectal cancer

目錄
謝 誌 ----------------------------------------------------------------------------- I
中文摘要---------------------------------------------------------------------------- Ⅱ
英文摘要-----------------------------------------------------------------------------Ⅲ
第一章 緒論
第一節 大腸直腸癌 --------------------------------------------------------- 1
第二節 大腸直腸癌之分期 ------------------------------------------------ 2
第三節 癌胚抗原 ------------------------------------------------------------ 4
第四節 大腸直腸癌的疾病發展------------------------------------------- 5
第五節 Wnt訊息路徑 ------------------------------------------------------ 6
第六節 Death-associated protein ------------------------------------------- 7
第七節 專一性上皮ETS轉錄因子---------------------------------------- 8
第二章 研究動機------------------------------------------------------------------ 12
第三章 研究材料及方法
第一節 細胞培養------------------------------------------------------------ 13
第二節 臨床組織檢體------------------------------------------------------ 14
第三節 西方墨點轉漬分析------------------------------------------------ 14
第四節 統計分析------------------------------------------------------------ 16
第四章 實驗流程------------------------------------------------------------------ 17
第五章 實驗結果------------------------------------------------------------------ 18
第一節 Daxx mRNA在正常組織與大腸直腸癌檢體組織的表現
--------------------------------------------------- 19
第二節 Daxx蛋白表現之T/N比值在不同Duke期別的分布趨勢
--------------------------------------------------- 19
第三節 分析手術前後CEA值的高低與臨床檢體中Daxx蛋白表現量
--------------------------------------------------- 20
第四節 分析EHF蛋白在不同結腸直腸癌細胞株當中的表現------------ 21
第五節 分析EHF蛋白在大腸直腸癌檢體組織之表現結果--------------- 21
第六節 分析臨床組織檢體中不同腫瘤侵犯深度之EHF表現量
------------------------------------------------------ 22
第六章 討論 ------------------------------------------------------------------ 23
第七章 結論--------------------------------------------------------------- 25
參考文獻 --------------------------------------------------------------------------- 37
附表一.大腸直腸癌期別腫瘤大小－淋巴轉移－遠處轉移, AJCC,8th
---------------------------------------------------------------------------------------- 40
























表目錄

表1分析整體個案的年齡、性別、Duke期別------------------------------- 28
表2個案Duke期別與Daxx蛋白表現之T/N比值的分布趨勢--------- 29
表3個案 CEA值(術前CEA)與Daxx蛋白表現T/N比值的分布---------------------------------------------------------------------------- 30
表4個案手術後CEA(CEA2)上升超過手術前CEA(CEA1)值之情況與
Daxx蛋白表現T/N比值的分布----------------------------------------- 31
表5分析臨床檢體中EHF蛋白的表現及T/N比值------------------------35
表6 EHF蛋白與腫瘤侵犯深度期別之相關性-----------------------------36



圖目錄

圖1研究策略----------------------------------------------------------------------- 26
圖2 Daxx mRNA在正常組織與大腸直腸癌檢體組織的表現------------ 27
圖3 Daxx靜默細胞株的建立--------------------------------------------------- 32
圖4 EHF與Daxx在不同Duke期別大腸直腸癌細胞株之表現結果--- 33
圖5 EHF在CRC配對檢體中的表現趨勢----------------------------------- 34

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