大腸直腸癌是一個預後較差的惡性腫瘤，通常在癌症初期症狀都不明顯，發現時大都屬於癌症晚期，且已有肝臟轉移的現象。CEA （Carcinoembryonic Antigen）可作為臨床上大腸直腸癌診斷鑑別工具，但是除了癌症之外，在腸道炎症中CEA數值也會偏高，所以找尋更好的生物標記以診斷結腸直腸癌是未來的重要方向。
Daxx在細胞核中扮演一個重要轉錄抑制者，實驗室先前的結果發現Daxx會抑制Wnt之轉錄活性，在結腸組織中明顯減少，透過microarray篩選出CD24以及EHF（Ets homologous factor）可能會受到Daxx蛋白表現所調控。有研究發現在多種惡性腫瘤中CD24會過度表現，增加癌細胞的侵襲力並可能縮短癌症病患的存活率，EHF則透過轉錄調控方式扮演腫瘤抑制的功能。因此在本研究論文中進一步偵測Daxx蛋白和β-catenin以及CD24蛋白或EHF蛋白在大腸直腸癌不同的癌症分期間之相關性。
首先收集106位大腸直腸癌病人檢體，分析病人手術前及手術後的CEA值與Daxx蛋白表現比較呈負相關，手術前≧ 5ng/ml 的CEA值有39位（佔45.1%），而Daxx＜0.86有58位（佔67.4%），因此Daxx蛋白表現有潛力彌補CEA的診斷限制。以西方墨點法分析各蛋白的表現差異，發現Daxx的蛋白與CD24蛋白以及EHF蛋白的表現都呈正相關性（P=0.041, P=0.017）；另外EHF蛋白表現量偏低者，在侵襲深度T3期有53位佔71.6%）；而在侵襲深度T4期有4位（佔100%）。也就是說EHF蛋白表現愈低，其癌細胞侵襲之深度亦增加（P＜0.05）。
在細胞實驗中利用Immunofluorscence確認Daxx蛋白、CD24蛋白以及β-catenin在細胞內的表現位置，發現在Daxx靜默的Hct116細胞中，CD24的表現明顯地由細胞膜轉位到細胞質。並發現Daxx蛋白表現降低，確實會增加該細胞株的生長速率以及移行能力。如此上述，Daxx蛋白及其調控路徑的建立和研究，可以讓我們更了解結腸直腸癌的形成機制，進而找出相關的標記如：CD24、EHF，可做為日後的研究方向。

ABSTRACT
Colorectal cancer (CRC) is one of the most malignancies in Taiwan. Early symptom is not easy to diagnosis, but lately stage almost has metastasis and worse prognosis. CEA level is considered as a predictive factor in colorectal cancer patients. But elevated CEA was found in other malignancies and inflammation. So, it is very important to found up another biological tumor marker to instead of limited CEA.
In our previous study, Daxx affected Tcf4 activity and regulated cell proliferation and differentiation in colon cells. CD24 and EHF were candidate genes regulated by Daxx expressing which were identified by gene microarray. Because of higher CD24 expression is associated with metastasis and shorter survival in various malignant tumors. On the other hand, Individual EHF can inhibit carcinogenesis by transcriptional regulation. It is our interesting to understand correlation between Daxx, β-catenin and CD24 or EHF in CRC carcinogenesis.
First, in clinical 106 paired CRC tissues analysis, a negative correlation was observed between CEA and Daxx. There were 39 patients (45.3%) with elevated CEA, but 58 patients (67.4%) with decreased Daxx expression, so Daxx may have diagnostic potential instead to CEA. In protein analysis, positive correlation was observed between CD24 or EHF with Daxx by Pearson’s correlation coefficient test (p=0.041, p=0.017). Reduction of EHF expression was relative to depth of invasion (p<0.05).
In cell lines, immunofluorescence analysis was performed, and CD24 was translocated from membrane to cytosol in Daxx knockdown stabled Hct116. On the other hand, cellular growth and cellular migration were increased rather than control cells. With our investing- ation above, Daxx may play a role to influence a prognostic factor, CD24 or EHF in CRC model, and further evidences will be investigated .

縮寫(Abbreviation) --------------------------------------------------------------------------1
壹、緒論
(一)大腸直腸癌(Colorectal cancer, CRC)------------------------------------------ ----2
(二)大腸直腸癌之分期----------------------------------------------------------------- ---3
(三)癌胚抗原 (Carcinoembryonic Antigen, CEA) ------------------------------ ----4
(四) Wnt訊息路徑(Wnt signaling pathway)--------------------------------------- --5
(五) Daxx (Death associated protein)------------------------------------------------ -7
(六)CD24------------------------------------------------------------------------------- -------9
(七)專一性上皮ETS轉錄因子-3(Epithelial-specific ETS, ESE-3)---------------11
貳、研究動機--------------------------------------------------------------------------------------13
叁、研究材料及方法
(一)細胞培養 (cell culture)------------------------------------------------------------14
(二)臨床組織------------------------------------------------------------------------------15
(三)西方墨點轉漬分析-----------------------------------------------------------------16
(四)免疫螢光染色( Immuno-fluorescence stain )-------------------------------18
(五)細胞移行試驗( Cell Migration assay )-----------------------------------------19
(六)統計分析(Statistical Analysis)---------------------------------------------------20
肆、實驗流程-------------------------------------------------------------------------------------21
伍、實驗結果
(一)探討Daxx是否能取代CEA之診斷於大腸直腸癌檢體---------------------22
(二)探討於大腸直腸癌組織檢體中Daxx與Wnt活性之相關-----------------23
(三)探討於大腸直腸癌中Daxx蛋白對CD24蛋白表現之影響
(1) CD24蛋白在大腸直腸癌組織檢體之表現-------------------------------24
(2)比較Daxx的蛋白表現與CD24蛋白表現相-------------------------------25
(四)探討於大腸直腸癌中Daxx蛋白對EHF蛋白表現之影響
(1) EHF蛋白在大腸直腸癌組織檢體之表現--------------------------------25
(2)比較Daxx的蛋白表現與EHF蛋白表現相關性--------------------------26
(3)比較CD24的蛋白表現與EHF蛋白表現相關性--------------------------27
(五)探討shLuci細胞株和shDaxx之Daxx穩定靜默細胞株蛋白之生物功能
(1)檢測CD24在不同細胞株的表現--------------------------------------------27
(2)利用免疫螢光染色於大腸直腸癌細胞株鑑定CD24和其他蛋白
在細胞內之表現位置 -------------------------------------------------------27
(3)不同細胞株經由shDaxx之Daxx穩定靜默處理後之細胞生
長變化-----------------------------------------------------------------------------29
(4)Daxx蛋白會減少對人類大腸直腸癌細胞株Hct116之移行能力
--------------------------------------------------------------------------------------30
陸、討論
(一)探討Daxx是否能取代CEA之診斷於大腸直---------------------------------31
(二)探討於大腸直腸癌組織檢體中Wnt訊息傳遞途徑之活性-------------32
(三)探討於大腸直腸癌中Daxx蛋白對CD24蛋白表現之影響---------------33
(四)探討於大腸直腸癌中Daxx蛋白對EHF蛋白表現之影響-----------------34
(五)探討於大腸直腸癌中CD24蛋白在細胞內之表現位置------------------34
(六)探討shLuci細胞株和shDaxx之Daxx穩定靜默細胞株蛋白之生物功能----------------------------------------------------------------------------------------35
柒、結論------------------------------------------------------------------------------------38
捌、結果圖與附圖-----------------------------------------------------------------------------39
Fig.1A、------------------------------------------------------------------------------------39
Fig.1B、Daxx蛋白表現與手術前CEA值和手術後CEA值相互比較--------40
Fig.1C、Daxx蛋白表現與手術後CEA值相較後可補足CEA之限制--------40
Fig.2、各蛋白在大腸直腸癌檢體中的表現趨勢------------------------------41
Table.1、Daxx的蛋白表現與β-catenin蛋白表現呈明顯顯著-----------42
Table.2、Daxx的蛋白表現與CD24蛋白表現呈明顯顯著-------------------43
Fig.3、Daxx的蛋白表現與CD24蛋白表現呈正相關性-----------------------44
Table.3、Daxx的蛋白表現與EHF蛋白表現無明顯顯著----------------------45
Fig.4、Daxx的蛋白表現與EHF蛋白表現呈正相關性-------------------------46
Fig.5、CD24的蛋白表現與EHF蛋白表現呈正相關性-------------------------47
Fig.6、在人類細胞株中檢測各蛋白的表現-------------------------------------48
Fig.7A、利用免疫螢光染色觀察人類大腸直腸癌細胞株Hct116細胞內
Daxx的表現---------------------------------------------------------------------49
Fig.7B、利用免疫螢光染色觀察人類大腸直腸癌細胞株Hct116細胞內
β-catenin的表現---------------------------------------------------------------50
Fig.7C、利用免疫螢光染色觀察人類大腸直腸癌細胞株Hct116細胞內
CD24的表現-------------------------------------------------------------------51
Fig.7D、利用免疫螢光染色觀察人類大腸直腸癌細胞株Hct116細胞內
p53的表現---------------------------------------------------------------------52
Fig.8A、免疫螢光染色觀察人類腎臟上皮細胞株293T細胞內Daxx的
表現-----------------------------------------------------------------------------53
Fig.8B、免疫螢光染色觀察人類腎臟上皮細胞株293T細胞內β-catenin的
表現-----------------------------------------------------------------------------54
Fig.8C、免疫螢光染色觀察人類腎臟上皮細胞株293T細胞內CD24的
表現-----------------------------------------------------------------------------55
Fig.8D、免疫螢光染色觀察人類腎臟上皮細胞株293T細胞內p53的
表現-----------------------------------------------------------------------------56
Fig9、Daxx表現減少對人類腎臟上皮細胞株293T的生長沒有明顯影響------------------------------------------------------------------------------------57
Fig10、Daxx表現減少會促進人類大腸直腸癌細胞株Hct116的生長----58
Fig.11、Daxx表現減少會促進人類大腸直腸癌細胞株Hct116多於人類腎
臟上皮細胞株之移行能力------------------------------------------------59
Fig.12、Daxx蛋白的再表現會減少Hct116Daxx靜默穩定細胞株之移行
能力-----------------------------------------------------------------------------61
玖、參考文獻-----------------------------------------------------------------------------------65

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