細胞週期蛋白D1 mRNA在小鼠胚胎及成體幹細胞和腫瘤細胞中的表現及其受多能性相關因子影響之探討

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廖家男(Chia-nan Liao) 查詢紙本館藏

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系統生物與生物資訊研究所

論文名稱

細胞週期蛋白D1 mRNA在小鼠胚胎及成體幹細胞和腫瘤細胞中的表現及其受多能性相關因子影響之探討

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摘要(中)

胚胎幹細胞具有自我更新及可分化為來自三個胚層組織約200多種細胞的能力。因胚胎幹細胞經培養與體內或體外誘導分化為成體細胞後，呈現人類生物及生理上之組織細胞特性與功能，故其不但具有用於基礎醫學如胚胎發育與疾病細胞機制探討之意義，而且在臨床醫療如藥物篩選與移植醫療方面也具有應用之潛力。但是，因胚胎幹細胞在來源及應用方面受限於相關倫理等問題，近年來由此而發展出誘導多能性幹細胞。利用基因轉殖技術導入Oct3/4、Sox2、Klf4與c-Myc因子，而使細胞有效地倒程式化而重組誘導成為具有與胚胎幹細胞相似特性之誘導多能性幹細胞。已知細胞週期蛋白D1是調控細胞週期的重要因子之一，其於哺乳動物細胞內可與其它調控次單元組成全脢，並於癌症細胞內會過度表現而影響到轉錄、轉譯與蛋白質穩定性等因素。本研究探討細胞週期蛋白D1於幹細胞內及細胞倒程式化過程中所扮演的角色，從而進一步探討誘導多能性幹細胞在分子層面上與細胞週期蛋白D1的關聯性。我們的結果發現細胞週期蛋白D1不但於小鼠誘導多能性幹細胞中明顯被抑制，而且此抑制現象也發生於個別全能性相關因子轉殖後之小鼠纖維母細胞中。從我們實驗結果得知，細胞重組或重組之基因皆會抑制細胞週期蛋白D1的表現，此結果顯示細胞週期蛋白D1可能在細胞倒程式化或分化過程中扮演重要的角色。

摘要(英)

Embryonic stem cells (ESCs) have ability of self-renewal and differentiation to about 200 types of cells over from the three germ layers. Because somatic cells that appear the property of biological and physiological functions can be derived by ESCs through culturing and inducing for differentiation in vivo and in vitro, the study of ESCs has not only significance for the basic researches in embryo development and cell mechanism of pathology, but it has the potentiality for the biomedical application in drug screening and tissue transplantation. However, the clinical application of ESCs has been restricted by reasons of ethical issue and deriving cell source. Thus the techniques of induced pluripotent stem cells (iPSCs) have been developed recently. Basically, a cell reprogramming was occurred by transfection of Oct3/4, Sox2, Klf4 and c-Myc factors into the somatic cells. This reprogramming induces somatic cells effectively to iPSCs that cells exhibited pluripotency as well as ESCs. It has been known that Cyclin D1 is composed the regulatory subunit of a dimeric holoenzyme in mammalian cells. Cyclin D1 is frequently overexpressed in cancers and its overexpression also has been implicated to many factors including increased transcription, translation, and protein stability. In this study, we discuss what a role of cyclin-D1 is in the stem cells and the further correlation about cyclin-D1 with cell reprogramming in the molecular level. In our results, we found that cyclin-D1 was not only inhibited distinctly from the early stage of reprogramming in mouse ESCs, but also down-regulated by respectively tranfecting 4 defined pluripotent genes. Our data suggest that cyclin-D1 plays possibly an important role in cell differentiation and reprogramming.

關鍵字(中)

★ 細胞週期蛋白D1
★ 幹細胞
★ 多能性相關因子

關鍵字(英)

論文目次

摘要 i
Abstract ii
致謝 iii
圖目錄 vii
表目錄 viii
中英名詞對照表 ix
第一章緒論 1
1-1 前言 1
1-2 幹細胞研究與應用 3
1-3 誘導多能性幹細胞之發明及研究的發展 5
1-4 細胞週期蛋白D1 之研究與發展 7
第二章實驗材料與方法 8
2-1 細胞製備 8
2-1-1 培養皿Gelatin Coating處理 8
2-1-2 小鼠胚胎纖維母細胞之製備 8
2-1-3 餵養細胞之製備 9
2-1-4 小鼠骨髓細胞之製備 9
2-2 細胞培養 10
2-2-1 小鼠胚胎纖維母細胞之培養與繼代 10
2-2-2 小鼠胚胎幹細胞之培養與繼代 10
2-2-3 小鼠誘導多能性幹細胞之培養與繼代 11
2-2-4 293FT 細胞之培養與繼代 12
2-2-5 CCRF S-180 Ⅱ小鼠腫瘤細胞株之培養與繼代 13
2-2-6 小鼠骨髓細胞之培養與繼代 13
2-3 細胞冷凍與解凍 14
2-3-1 幹細胞冷凍與解凍 14
2-3-2 成體、腫瘤細胞之冷凍與解凍 14
2-4 螢光染色 15
2-4-1 貼附型細胞免疫螢光染色 15
2-4-2 流式細胞螢光染色 16
2-5 基因轉殖 17
2-5-1 反轉錄病毒製成 17
2-5-2 反轉錄病毒轉染 18
2-6 基因表現 19
2-6-1 RNA萃取 19
2-6-2 逆轉錄聚合酶連鎖反應 20
第三章結果 24
3-1 細胞型態 24
3-1-1 小鼠胚胎纖維母細胞與腫瘤細胞 24
3-1-2 小鼠骨髓細胞 25
3-1-3 小鼠胚胎幹細胞與誘導多能性幹細胞 26
3-2 細胞特殊生物蛋白質標記之鑑定 27
3-2-1 小鼠胚胎幹細胞之生物標記表現 28
3-2-2 小鼠誘導多能性幹細胞之生物標記表現 29
3-2-3 小鼠骨髓細胞流式細胞儀實驗結果 30
3-3 基因表現 31
3-3-1 胚胎幹細胞相關生物因子之基因表現 31
3-3-2 細胞週期蛋白D1之表現 32
3-3-3 細胞週期蛋白D1與全能性相關個別因子之關係 33
第四章討論 34
4-1 細胞週期蛋白D1之功能 34
4-2 幹細胞的細胞週期 36
4-3 細胞週期蛋白D1與細胞倒程式化關聯性之探討 38
第五章總結 39
第六章參考文獻 40

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指導教授

凌慶東(Qing-Dong Ling)

審核日期

2012-12-19

推文