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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/17851

Title: 上皮細胞黏附因子與上皮型鈣黏附蛋白於再編程老鼠成體細胞為誘導型全能性幹細胞之表現
Expression of Epithelial cell adhesion molecule and Epithelial-cadherin in reprogramming mouse somatic cells into induced pluripotent stem cells (iPSCs)
Authors: Wen-Chih Lee
李文志
Contributors: NTOU:Institute of Bioscience and Biotechnology
國立臺灣海洋大學:生物科技研究所
Keywords: 上皮細胞黏附因子;上皮型鈣黏附蛋白;誘導型全能性幹細胞;再編程
EpCAM;E-cadherin;iPSCs;reprogram
Date: 2010
Issue Date: 2011-07-04
Abstract: 以反轉錄病毒或慢病毒轉導Oct4、Sox2、Klf4和c-Myc四個基因至老鼠或人類之成體細胞,可產生誘導型全能性幹細胞 (induced pluripotent stem cells;iPSCs) ,使得患者特異性 (patient-specific) 之iPSCs應用於再生醫療變為可能。但就大多篩選具全能特性之iPSCs之技術中,是於全能性基因NANOG與Oct4之所在染色體位置預先插入報導式基因 (reporter genes) ,如用於臨床上更是一大阻礙。因此,以表面標的鑑定iPSCs之全能特性,亦可達到篩選之目的,並有助於解決上述之問題。我們以慢病毒分別轉導Oct4、Sox2、Klf4與n-Myc四個基因至老鼠胚胎纖維母細胞 (mouse embryonic fibroblast cells;MEFs) ,利用老鼠誘導型全能性幹細胞 (mouse induced pluripotent stem cells;miPSCs) 之產生過程,作為測試表面標的之平台。選擇已證實在胚胎幹細胞會表現上皮細胞黏附因子 (epithelial cell adhesion molecule;EpCAM) 與上皮型鈣黏附蛋白 (Epithelial-cadherin;E-cadherin) 做為初步測試之表面標的。經外觀挑選類老鼠胚胎幹細胞株 (mouse embryonic stem cell-like colony;mES-like colony) ,於不表現全能性基因NANOG與內源性基因Oct4和n-Myc之細胞株中,我們發現鹼性磷酸酶 (alkaline phosphatase) 與階段性特異胚胎抗原1 (stage-specific embryonic antigen 1;SSEA1) 已開始表現,因而無法鑑定經再編程 (reprogram) 之細胞開始具全能之特性。雖EpCAM與E-cadherin之表現也與全能性基因NANOG無直接關係,但我們於結果中發現,似乎可判斷此再編程之細胞是處於內源性基因Oct4與n-Myc開始表現之階段。因此,以EpCAM與E-cadherin做為篩選之標的,應可提高篩得具全能特性之iPSCs之機率。
Induced pluripotent stem cells (iPSCs) have been generated from mouse and human somatic cells through Oct4, Sox2, Klf4, and c-Myc gene transduction by retroviruses or lentiviruses. The development of iPSCs improves the possibility for the production of patient-specific pluripotent stem cells in regenerative medicine. However, in most of iPSCs screening techniques, the requirement of insertion of reporter genes in NANOG and/or Oct4 chromosome locus has restricted the clinical application of iPSCs. Therefore, characterizing pluripotency of iPSCs by surface markers could help us to screen fully reprogrammed iPSCs and prevent previous problems. In this study, we introduced lentiviral Oct4, Sox2, Klf4, and n-Myc into mouse embryonic fibroblast cells (MEFs). We used it as a model to develop a mouse induced pluripotent stem cells (miPSCs) screening platform during miPSCs formation. First, in mouse embryonic stem cell-like colony (mES-like colony) picked in accordance with their morphology, we observed the expression of alkaline phosphatase and stage-specific embryonic antigen 1 (SSEA1) without the expression of endogenous NANOG, Oct4 and n-Myc, suggesting that alkaline phosphatase or SSEA1 might not be appropriate markers for fully reprogrammed miPSCs. Next, we chose Epithelial Cell Adhesion Molecule (EpCAM) and Epithelial-cadherin (E-cadherin) as candidate surface markers for screening fully reprogrammed miPSCs. Although EpCAM or E-cadherin were reportedly not correlate with the pluripotent gene—NANOG, we found that EpCAM and E-cadherin-expressed cells seemed under the stage which endogenous Oct4 and n-Myc just about to express. In conclusion, EpCAM and E-cadherin could be selection surface markers that increase the possibility to obtain fully reprogrammed iPSCs.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M96360029
http://ntour.ntou.edu.tw/ir/handle/987654321/17851
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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