臺灣博碩士論文加值系統

上皮細胞黏附因子(Epithelial cell adhesion molecule，EpCAM)是一種細胞表面蛋白分子，在許多的癌症細胞表現，並具有引發癌症的潛能。其被蛋白脢剪切後，釋放其胞內部份EpICD (EpCAM intracellular domain)，進而活化 c-Myc (C terminal myelocytomatosis oncogene) 的表現。此外，EpCAM與緊密連結蛋白中的CLDN-7 (Claudin-7) 所形成的複合體則會促進腫瘤形成與發育。我們觀察到在老鼠胚胎幹細胞(mouse embyronic stem cells，mES)與經由反程序化而形成之誘導型多分化潛能幹細胞(induced pluripotent stem cell，iPS)皆會表現EpCAM及CLDN-7。且EpCAM與CLDN-7會隨著反程序化的過程而逐漸活化表現。而大量表現EpCAM及EpICD則會提高誘導型多分化潛能幹細胞的形成效率，再者，CLDN-7可以協同EpCAM增進誘導型多分化潛能幹細胞的產率。反之，抑制EpCAM及CLDN-7的表現則會降低誘導型多分化潛能幹細胞的產率。然而，在已分化之體細胞(老鼠纖維母細胞)中單獨表現EpCAM或CLDN-7並無法活化多分化潛能基因之表現。我們的研究結果顯示，EpCAM可以正向調控反程序化的效率，但其調控機制則需更進一步的研究。

Epithelial cell adhesion molecule (EpCAM) is a cell adhesion molecule and a potential target for therapeutic antibodies. Recently, EpCAM has been shown to regulate tumor cell proliferation by its nucleocytoplasmic intracellular domain fragment (ICD). EpCAM intracellular domain (EpICD) activates C terminal myelocytomatosis oncogene (c-Myc) in nucleus by Wnt signal pathway. On the other hand, the complex of EpCAM and the tight junction protein claudin-7 (CLDN-7) would promote tumorigenicity and accelerates tumor growth. We observed that EpCAM and CLDN-7 express in both mouse embryonic stem cells and induced poluripotent stem cells. In this study we first discovered that the expressions of EpCAM and CLDN-7 would be activated when reprogramming mouse embryonic fibroblast cells into induced pluripotent stem cells. The efficiency of iPS reprogramming was enhanced by overexpressing EpCAM, CLDN-7 or both. Furthermore, silence of endogenous EpCAM or CLDN-7 resulted in the reduction of iPS reprogramming. However, ectopic expression of EpCAM and CLDN-7 in differential somatic cells such as mouse fibroblast cells had no effect on the activation of pluripotency-associated gene Nanog, Oct4, Sox2, c-Myc and KLF4. Our data show that EpCAM positively regulates iPS reprogramming, yet the detail mechanism remains unclear.

謝辭 ……………………………………………………………………… i
中文摘要 ………………………………………………………………… ii
英文摘要 …………………………………………………………………iii
縮寫表 …………………………………………………………………… x
第一章 緒論
一. 幹細胞（Stem Cells）……………………………………………1
1. 幹細胞的分類與特性
2. 研究胚胎幹細胞所面臨之問題
二. 誘導型多分化潛能幹細胞（induced pluripotent stem cells，iPS）
……………………………………………………………………5
1. 誘導型多分化潛能幹細胞之研究發展
2. 反程序化過程之基因表現關係
三. 表皮黏附因子（Epithelial cell adhesion molecule，EpCAM）及緊密結合蛋白Claundin-7（tight junction protein Claudin-7）…10
1. 蛋白質特性
2. 反應機制
四. 研究動機…………………………………………………………14

第二章 材料與實驗方法
一. 細胞培養…………………………………………………………15
1. 老鼠纖維母細胞培養
2. 老鼠胚胎幹細胞培養
二. 質體製備（plasmid）……………………………………………18
1. 小量製備質體
2. 中量製備質體
三. 反轉錄病毒製備（retrovirus）……………………………………22
四. 誘導型多分化潛能幹細胞製備（miPSCs）……………………24
五. 鹼性磷酸酶染色（AP stain）……………………………………25
六. 西方點墨法（Western Blot）……………………………………26
七. 免疫螢光染色（IF Stain）………………………………………30
八. RNA萃取 ………………………………………………………32
九. 反轉錄聚合酶連鎖反應（Reverse Transcription Polymerase Chain Reaction） ………………………………………………………34
十. 即時聚合脢連鎖反應（Real-Time Polymerase chain reaction）…35
十一. 聚合脢連鎖反應（Polymerase chain reaction） …………37
十二. 洋菜膠體電泳（Agarose Gel Electrophoresis） …………38

第三章 結果
一. 檢測內源性EpCAM與CLDN-7在老鼠胚胎幹細胞及老鼠誘導型多分化潛能幹細胞中的表現情形……………………………39
二. 分析 EpCAM、CLDN-7、Nanog、Oct4 及Sox2於老鼠纖維母細胞反程序化為誘導型多分化潛能幹細胞過程中之表現情形
……………………………………………………………………40
三. 檢測於老鼠纖維母細胞反程序化為誘導型多分化潛能幹細胞過程中抑制EpCAM或CLDN-7的表現是否對反程序化產生影響
……………………………………………………………………42
四. 檢測於老鼠纖維母細胞反程序化為誘導型多分化潛能幹細胞過程中過量表現EpCAM、CLDN-7或EpICD 是否對反程序化產生影響……………………………………………………………44
五. 檢測於終端分化細胞（老鼠纖維母細胞）中單獨過量表現EpCAM、CLDN-7或EpICD 是否影響多分化潛能基因的表現
……………………………………………………………………47
第四章 討論 …………………………………………………………… 49
第五章 參考文獻 ……………………………………………………… 54

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