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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林泰元 | |
| dc.contributor.author | Yu-Chi Wang | en |
| dc.contributor.author | 王育婍 | zh_TW |
| dc.date.accessioned | 2021-06-08T04:19:00Z | - |
| dc.date.copyright | 2010-09-13 | |
| dc.date.issued | 2010 | |
| dc.date.submitted | 2010-07-24 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22489 | - |
| dc.description.abstract | 成體幹細胞分佈於各種組織器官，負責維持及修護特定組織之完整性。目前已鑑定出至少11種的肺部成體幹細胞，然而大多數的肺部成體幹細胞無法進行持續且大量的細胞培養，以提供肺部細胞研究之平台。先前，我們發展出一套無血清培養之小鼠Oct4+肺部上幹/前驅細胞。這群細胞表現多功能性且具有對嚴重急性呼吸道症候群（SARS）冠狀病毒、A型流感病毒以及單純疱疹病毒第一型之感染性。上述發現高度支持小鼠Oct4+肺部上皮幹/前驅細胞在肺部疾病之研究與臨
床應用之潛力，但小鼠Oct4+肺部上皮幹/前驅細胞也同樣面臨難以大量培養的限制。因此，本篇藉由體細胞再程序化技術（轉導入Oct4, Sox2, Klf4及c-Myc）誘導出不朽化之小鼠Oct4+肺部上皮幹/前驅細胞株。結果顯示不朽化之小鼠Oct4+肺部上皮幹/前驅細胞維持與初級小鼠Oct4+肺部上皮幹/前驅細胞相似的表現型，此細胞株同時顯示高度的端粒酶活性，也能夠分化成第一型及第二型肺泡細胞。此外，這株細胞處在高度增生的狀態，且能夠形成畸胎瘤，暗示了再程序化之過程不只誘導了肺部幹/前驅細胞之多功能性及自我更新能力，也可能將正常的肺部幹/前驅細胞導向腫瘤細胞的命運。 本研究證實利用體細胞再程序化能夠誘導出不朽化之小鼠Oct4+肺部上皮幹/前驅細胞，並能將此細胞株應用於病毒感染之研究。 | zh_TW |
| dc.description.abstract | Adult stem cells are distributed in many tissues and are responsible for regeneration and repair of specific tissues. Although more than 11 types of lung adult stem cells have been identified, few of them can be efficiently isolated and expanded in vitro. We have previously developed a serum-free mouse Oct4＋ pulmonary stem/progenitor cell (mPSC) culture system. These cells were multipotent and susceptive to SARS coronavirus, influenza A virus and Herpes Simplex virus type 1, suggesting their potential in basic research and clinic application. However, mPSCs could not be expanded in a large scale. Here, we used somatic reprogramming (i.e. transducing Oct4, Sox2, Klf4, and c-Myc into somatic cells) to generate an immortal mPSC (imPSC) line and maintain their multipotency. Our data demonstrate that the phenotype of imPSCs was comparable to which of primary mPSCs. Furthermore, imPSCs showed high telomerase activity and the capability of pulmonary differentiation. Importantly, they displayed vigorous proliferation and teratoma formation, suggesting that the cancerous transition of normal mPSCs could be triggered and facilitated by somatic reprogramming. Our work proposes that imPSCs could be generated by somatic reprogramming and served as a feasible line for the design of experimental and therapeutic approaches in virus infection and respiratory diseases. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T04:19:00Z (GMT). No. of bitstreams: 1 ntu-99-R97443011-1.pdf: 50136861 bytes, checksum: 0be34230503909dc33e97f962700d802 (MD5) Previous issue date: 2010 | en |
| dc.description.tableofcontents | 誌謝....................................................iii
Abbreviation.............................................iv 中文摘要..................................................v Abstract.................................................vi Chapter 1 Introduction....................................1 1.1. Pulmonary stem/progenitor cells......................2 1.1.1. Organization of pulmonary epithelium...............2 1.1.2. Stem/progenitor cells in pulmonary epithelium......2 1.2. Immortalization of primary somatic cells.............4 1.3. Transcription factors mediated somatic reprogramming.5 1.3.1. Generation of induced pluripotent stem cells.......5 1.3.2. Transcription factors for generation of iPSCs......6 1.3.3. Choice of cell types...............................8 1.4. Motivation...........................................9 1.5. Aim.................................................10 Chapter 2 Materials and methods..........................11 2.1. Cell culture........................................12 2.1.1. Primary culture of mouse pulmonary stem/progenitor cells....................................................12 2.1.2. Culture of Plat-E cells...........................13 2.1.3. Culture of immortalized pulmonary stem/progenitor cells....................................................13 2.2. Retrovirus production and infection.................14 2.3. Generation of immortalized pulmonary stem/progenitor cells....................................................15 2.3.1. Transduction of transcription factors into primary pulmonary stem/progenitor cells..........................15 2.3.2. Induction assay...................................15 2.4. Soft agar assay.....................................16 2.5. Magnetic-activated cell sorting.....................16 2.6. Flow cytometry......................................17 2.7. RNA isolation.......................................18 2.8. Reverse transcription and real time-quantitative PCR......................................................18 2.9. Alkaline phosphatase stainin........................19 2.10. Immunofluorescence staining........................19 2.11. Western blotting...................................20 2.12. Cell growth curve..................................20 2.13. Detection of telomerase activity................... 21 2.14. In vitro differentiation ...................... 21 2.15. Teratoma formation..............................22 Chapter 3 Results .................................. 23 List of figures and tables ...........................24 3.1. Identification of mPSCs ............................ 25 3.2. Optimization of retroviral transduction............. 26 3.3. Effect of individual factors on reprogramming of mPSCs........................... 26 3.4. Determination of optimal conditions for reprogramming and maintaining mPSCs ................. 27 3.5. Isolation of Sca1+ mPSCs ..........................29 3.6. Immortalization of primary mPSCs ....................30 3.7. Characterization of imPSCs ......................31 3.8. Expression of retroviral transgenes in imPSCs ............................................ 32 3.9. Clonogenic capability of imPSCs .....................33 3.10. In vitro differentiation of imPSCs..................33 3.11. Teratoma formation of imPSCs ...................... 35 Chapter 4 Discussion.................................... 36 4.1. Roles of transcription factors in reprogramming primary mPSCs............... 37 4.2. Effects of micro environment on the induction efficiency of imPSCs......... 38 4.3. Immortalization of primary mPSCs by tansducing reprogramming factors.......... 39 4.4. Teratoma formation of imPSCs.................. 41 4.5. Pluripotency of imPSCs ..................... 43 Chapter 5 Conclusion................................... 45 Chapter 6 References .................................. 48 Figures and tables....................................... 55 | |
| dc.language.iso | en | |
| dc.title | 小鼠肺部上皮幹/前驅細胞再程序化之研究 | zh_TW |
| dc.title | The Study of Somatic Reprogramming of Mouse Pulmonary Stem/Progenitor Cells | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 98-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 楊泮池,陳惠文,黃彥華,郭紘志 | |
| dc.subject.keyword | 肺部上皮幹/前驅細胞,體細胞再程序化,細胞不朽化, | zh_TW |
| dc.subject.keyword | Mouse pulmonary stem/progenitor cells,somatic reprogramming,immortalization, | en |
| dc.relation.page | 85 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2010-07-26 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 藥理學研究所 | zh_TW |
| 顯示於系所單位： | 藥理學科所 | |
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