非整倍性和染色體不穩定（CIN）是人類癌症中常見的異常。有絲分裂紡錘體檢查點的改變可能導致這些現象。為了探討染色體不穩定和結腸直腸癌之間的相關性作為診斷或預後標誌物和形成新的治療策略的可能性。在本篇論文中，使用生物資訊資料分析(infromatics-base assay)做為基礎的分析，找出大腸癌中58個和染色體分離的相關基因。其中三個基因，NDC80，Nuf2和SPC25屬於NDC80複合體，其複合體在染色體分離具有關鍵作用。為了了解NDC80在結腸癌組織的表達，以結腸直腸癌組織切片針對NDC80蛋白質進行免疫組織化學染色。141例結腸直腸癌組織中有90例具有較高的NDC80表達，然而相鄰的正常組織未檢測到NDC80表達。為了闡明NDC80在結腸直腸癌腫瘤進展的重要意義，我們與主要的臨床特徵進行分析。NDC80高表達與組織病理學中T-分期（T2〜T3，P = 0.002），晚期腫瘤（第II期及第III期，P <0.001）密切相關。結直腸癌中，第II和III期大腸癌患者，NDC80高表達有較低的12年存活率。為了進一步了解NDC80在結腸癌細胞中的作用和基因組不穩定性，我們用RAPD-PCR檢測，分析非整倍性和染色體不穩定（CIN）是人類癌症中常見的異常。有絲分裂紡錘體檢查點的改變可能導致這些現象。為了探討染色體不穩定和結腸直腸癌之間的相關性作為診斷或預後標誌物和形成新的治療策略的可能性。在本篇論文中，使用生物資訊資料分析(infromatics-base assay)做為基礎的分析，找出大腸癌中58個和染色體分離的相關基因。其中三個基因，NDC80，Nuf2和SPC25屬於NDC80複合體，其複合體在染色體分離具有關鍵作用。為了了解NDC80在結腸癌組織的表達，以結腸直腸癌組織切片針對NDC80蛋白質進行免疫組織化學染色。141例結腸直腸癌組織中有90例具有較高的NDC80表達，然而相鄰的正常組織未檢測到NDC80表達。為了闡明NDC80在結腸直腸癌腫瘤進展的重要意義，我們與主要的臨床特徵進行分析。NDC80高表達與組織病理學中T-分期（T2〜T3，P = 0.002），晚期腫瘤（第II期及第III期，P <0.001）密切相關。結直腸癌中，第II和III期大腸癌患者，NDC80高表達有較低的12年存活率。為了進一步了解NDC80在結腸癌細胞中的作用和基因組不穩定性，我們用RAPD-PCR檢測，分析NDC80表現量在DLD-1（二倍體/ CIN -）和HT-29（非整倍體/ CIN+）結腸癌細胞的基因組不穩定性相關性。結果顯示HT-29細胞與對照組ARPE19細胞相比，有較高的基因組改變。此外，使用FFPE CRC組織切片中抽取DNA樣本，比較高NDC80基因表達和低NDC80表達的差異。結果說明了NDC80表達高低與基因組改變是相關的。為了研究NDC80在結腸癌細胞生長的作用，NDC80蛋白表達抑制的大腸癌細胞株。使用CellTiter測定癌細胞生長率和集落形成試驗(Clonogenic assay)，結果HT-29細胞減少50％，但在DLD-1細胞減少70％以上。形態學檢查在細胞核顯示，轉染NDC80 RNAi會導致多核細胞，HT-29細胞只有5倍上升，但DLD-1細胞上升13倍。使用類流式影像(Image-flow)細胞測定法，在HT-29結腸癌細胞knockdownNDC80會增加sub G1階段和G2 / M期。此外，我們分析了一些染色體分離和染色體不穩定（CIN）相關的基因，我們可以發現細胞週期素B1蛋白下調，separase和securin分別上調在HT-29細胞，但不是在DLD-1細胞。這些結果表明抑制NDC80基因表達在結腸癌細胞可能會擾亂的染色體分離相關基因的表達和增加基因組不穩定性導致癌細胞死亡。

Aneuploidy and chromosomal instability (CIN) are common abnormalities in human cancer. Alterations of the mitotic spindle checkpoint are likely to contribute to these phenotypes. To elucidate the correlation between chromosomal instability and colorectal cancer might serve as diagnostic or prognostic markers and form the basis for novel therapeutic strategies. In this study, we used aninfromatics-base analysis to figure out 58 chromosome segregation genes in CRC. Three of 58 chromosome segregation genes, NDC80, Nuf2 and SPC25 belong to the NDC80 complex, which complex play a critical role in chromosome segregation. In order to understand the NDC80 expression in colon cancer tissue, we performed the immunohistochemistry stain for NDC80 protein using the colorectal cancer tissue section. 90 out of 141 CRCs had high NDC80 expression, however NDC80 expression was not detectable in adjacent normal part. To elucidate the significance of NDC80 in the tumor progression of CRC, we correlated theNDC80 protein expression with the major clinicopathological features. Histopathologically, NDC80 high-expression closely correlated with high-pathological T-staging (T2~T3, p=0.002), and high-stage tumors (stages II, III, p<0.001). CRCs with NDC80 high-expression had lower 12-year survival than those without the high-expression in stage II and III CRC patients. To further elucidate the role of NDC80 and genomic instability in colon cancer cells, we used RAPD-PCR assay to investigate the correlation of NDC80 expression level and genomic instability in DLD-1 (diploidy/CIN -) and HT-29 (aneuploidy/ CIN+) colon cancer cells. Therewere higher genetic alterations inHT-29 cell by compared with the reference ARPE19 cell. Furthermore, usingthe DNA samples extracted from FFPE CRC tissue sections, there were differential genetic alterationpatern between the high-NDC80 expression and low-NDC80 expression.These results indicated the NDC80 expression level should be correlated to the genetic alteration. To investigate the role of NDC80 in colon cancer cell growth, NDC80 protein expression was knockdown in CRC cells. The cancer cell growth rates were decreased 50% in HT-29 cells but 70% in DLD-1 cells using the celltitler assay and clonogenic assay. Morphological examination in cell nucleus revealed that transfection with the NDC80 RNAi oligo lead to multi-nucleated cells, almost 5 folds rise in HT-29cell but 13 folds rise in DLD-1 cell. Using the Image flow-cytometry assay, Down-regulation of NDC80 increased the sub-G1 phase in both colon cancer cells and increased the G2/M phase in HT-29 cells. Moreover, we followed up some chromosome segregation related genes which also contributed to the chromo-somal instability (CIN), we could find the cyclin B1 protein was downregulation, sepa-rase and securin were up regulation in HT-29 cells but not in DLD-1 cell. These results indicate knockdown NDC80 gene expression in colon cancer cell may disrupt the ex-pression of chromosome segregation related genes and increase genomic instability than lead to cancer cell death.

壹、【緒論】(Introduction)
1.1研究背景 1
1.2研究目的 4
貳、【材料與方法】(Materials and Methods)
2.1:臨床檢體收集 5
2.2試劑(Reagent)和實驗套組(Kit) 5
2.3:免疫組織化學染色(Immunohistochemistry)及判讀 6
2.4:細胞培養及siRNA降解作用 6
2.5:細胞同步(Ｃell synchronization) 7
2.6反轉錄-聚合酶鏈鎖反應(RT-PCR) 7
2.7隨機擴增多態性去氧核醣核酸(Random amplified polymorphic DNA ) RAPD-PCR 8
2.8細胞增殖分析(Celltiter assay) 10
2.9懸浮細胞分析(Spheroid assay) 10
2.10細胞群落形成分析法(Colony formation assay) 10
2.11免疫螢光染色(immunofluorescence staining) 10
2.12細胞塗片(Cytospin)及R415/DAPI stain 11
2.13:細胞週期分析(Cell cycle assay) 11
2.14西方墨點法(Western blot) 12
2.15統計方法 12
叁、【結果】(Results)
3.1資料庫分析 13
3.2免疫組織化學染色分析NDC80在組織中的表現量 13
3.3分析NDC80的表現與臨床表徵的相關性 14
3.4探討細胞週期與NDC80的關係 14
3.5闡明大腸癌中NDC80與基因不穩定性的關聯 14
3.6抑制NDC80表現會抑制體外腫瘤細胞生長及影響形成细胞群落 15
3.7細胞核形態學檢查，抑制NDC80會造成細胞產生多核現象 16
3.8 RAPD-PCR檢測經NDC80的RNAi處理後癌細胞的遺傳改變增加 17
3.9 類流式細胞影像測定法分析NDC80的降解下調對細胞週期的影響 17
3.10染色體分離相關基因在經NDC80的RNAi處理後的改變 17
肆、【討論】(Discussion) 18
伍、【圖示與圖表】(Figures and Tables) 23
陸、【參考文獻】(References) 52
柒、【附件】(Index) 58

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衛生統計，衛生福利部國民健康署，2007~2011.
衛生統計，大腸癌歷年死亡情形資料，衛生福利部統計處，2002~2014.