臺灣博碩士論文加值系統

腫瘤胚胎基因(Oncofetal Gene)是指在胚胎發育時期高度表現之基因，隨著個體年齡增長，其表現量降低或是不被表現。然而當個體患有惡性腫瘤時，腫瘤胚胎基因會再度被活化表達，而此一特性在臨床上能做為偵測癌症發生以及復發的指標。實驗室在先前已建立一套生物資訊系統，從表達序列標記片段(EST)資料庫中，尋找腫瘤胚胎候選基因，再藉由RT-PCR方式初步篩選出具有腫瘤胚胎表現型(Oncofetal Phenotype)基因進行研究。利用此生物資訊系統找出之Lin28B，已被證實為一個良好的循環腫瘤胚胎幹細胞標記(Circulating oncofetal tumor-stem-cell-like marker)，可辨識臨床檢體是否具有腫瘤幹細胞(Tumor stem cell)，並能針對病患進行轉移性復發及預後風險評估。
從上述生物資訊系統中找出另一個具有潛力之基因─C11orf41。C11orf41是一個已知核苷酸序列但生物功能未知的基因，經RT-PCR方式初步篩選後，發現在大多數腫瘤細胞株中均有表現，但僅在部分正常組織(胚胎時期腦部組織、成人腦部組織、睪丸和脊椎)中被偵測到，而在腫瘤-非腫瘤組織配對檢體中也有相似的結果，因此證實C11orf41為一個腫瘤胚胎基因。另外，在誘導性多功能幹細胞(iPS)及富有EpCAM (Stem Cell marker)之肝癌細胞中也能偵測到C11orf41的表現，進而提出C11orf41是一個具有潛力之腫瘤胚胎幹細胞標記的假設。
本篇研究首先構築C11orf41與EGFP及Myc epitope的融合蛋白，以反轉錄病毒感染方式建立Over-expressed C11orf41穩定細胞株，再藉由西方點墨法確認C11orf41蛋白質表現，並利用共軛焦顯微影像系統發現C11orf41會在細胞質中形成聚集蛋白(Aggregate Protein)。另外也建構了7 個C11orf41的截短蛋白(Truncated Protein)分別與EGFP及Myc epitope進行融合，以共軛焦顯微影像系統觀察，其中C11orf41(4519-5547b.p)和C11orf41(3988-5547b.p)在細胞核內會有聚集蛋白的形成。證明在C11orf41蛋白質C’端序列上除了具有聚集結構之外，可能還存在著核定位序列(NLS)，有助於C11orf41進入細胞核內參與基因之調控。因此，認為C11orf41可能是一個具有功能之蛋白質，但實際參與的作用機制仍待後續研究解謎。

Aim of the study was to validate the function of C11orf41 which was a potential oncofetal tumor-stem-cell marker. I have constructed the full-length of C11orf41 fused with EGFP or Myc epitope tag into pMSCV vector, and cloned 7 different truncated EGFP/ Myc epitope tag fusion proteins. To investigate the functions of C11orf41, Human HCC cell line, HepG2, was overexpressed C11orf41-EGFP by retrovirus infection and DNA transfection. The localization of C11orf41-EGFP fusion protein were accumulating on cell cytoplasm, and becoming insoluble in cells. In addition, COOH terminus proteins of C11orf41 were aggregated and translocated into nucleus. The study reveals that COOH terminus of C11orf41 may play an important role in protein’s function, but detailed mechanisms are still unknown.

中文摘要 I
英文延伸摘要 II
誌謝 V
目錄 VI
表目錄 X
圖目錄 XI
第一章 緒論 1
一、 Oncofetal Antigen 1
二、 Circulating tumor cell 2
三、 Tumor stem cell 2
四、 Bioinformatics 3
1. 表達序列標記片段 Expressed Sequence Tag 3
2. 生物資料庫(Bio-database)建立 4
3. 利用生物資訊尋找新穎的Oncofetal gene 5
4. 利用生物資訊尋找新穎的Wnt/-catenin標靶基因 5
5. 尋找新穎Oncofetal tumor-stem-cell –like gene 6
五、 C11orf41 information 7
1. C11orf41 fusion protein 7
2. Gene structure of C11orf41 8
六、 實驗目的 9
第二章 實驗材料與方法 10
一、 分子生物技術 10
1. 反轉錄聚合酶連鎖反應 (RT-PCR) 10
2. 構築質體的方法 12
3. C11orf41相關質體構築 14
二、 細胞培養程序 15
1. 實驗細胞株及試劑 15
2. 解凍細胞 16
3. 細胞繼代培養 16
4. 細胞數目測定 17
5. 細胞冷凍儲存 17
三、 細胞相關實驗 17
1. 細胞轉染 (Transfection) 17
2. 反轉錄病毒 (Retrovirus)製備與感染 18
3. 細胞增生實驗(WST-1 assay) 19
四、 西方點墨法 (Western blotting) 19
1. 試劑 19
2. 蛋白質樣品製備 21
3. 蛋白質濃度定量 21
4. 膠體電泳 22
5. 轉漬 (Transfer) 22
6. 轉漬後至ECL呈色 23
五、 免疫螢光染色 23
1. 樣品製備 23
2. 螢光顯微鏡觀察 24
第三章 結果 25
一、 確認生物資訊分析之結果 25
1. 證實C11orf41為腫瘤胚胎基因 25
2. 證明C11orf41可能為Wnt/-catenin標靶基因 25
3. 證實C11orf41表現與幹細胞特性(Stemness)相關 26
二、 C11orf41功能性探討 27
1. Overexpression 27
2. Knockdown 29
三、 C11orf41 truncated protein 29
1. C11orf41 truncated clone 29
2. Over-expressed C11orf41 truncated protein 30
四、 以癌症基因組圖譜(TCGA)資料庫預測C11orf41臨床意義 31
第四章 討論 32
一、 C11orf41 clone 32
1. C11orf41-Myc epitope 32
二、 Overexpression of C11orf41 32
1. Over-expression of C11orf41 protein 32
2. Over-expressed C11orf41 protein in cells 33
第五章 表 34
第六章 圖 40
第七章 參考文獻 58
第八章 附錄 62
附圖一、 利用生物資訊方法尋找新穎腫瘤胚胎幹細胞標記之流程圖 62
附圖二、 C11orf41在各種胎兒及正常成人組織中表現情形 63
附圖三、 以RT-PCR尋找可能的腫瘤胚胎幹細胞基因 64
附圖四、 C11orf41 在各種腫瘤-非腫瘤組織配對檢體中表現情形 65
附圖五、 C11orf41 在肺癌腫瘤-非腫瘤組織配對檢體中表現情形 66
附圖六、 C11orf41在肝癌腫瘤-非腫瘤組織配對檢體中表現情形 67
附圖七、 Lithium chloride對於C11orf41的mRNA表現之影響 68
附圖八、 利用染色質免疫沉澱法探討-catenin/TCF complex對C11orf41 promoter的結合 69
附圖九、 C11orf41 在iPS細胞中表現情形 70
附圖十、 以Real-time PCR偵測C11orf41在富集EpCAM之PLC/PRF/5細胞株中的表現量 71
附圖十一、 C11orf41 shRNA target sequence 72

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