(3.238.186.43) 您好!臺灣時間:2021/02/28 20:48
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:王育婍
研究生(外文):Yu-Chi Wang
論文名稱:小鼠肺部上皮幹/前驅細胞再程序化之研究
論文名稱(外文):The Study of Somatic Reprogramming of Mouse Pulmonary Stem/Progenitor Cells
指導教授:林泰元林泰元引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:藥理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:85
中文關鍵詞:肺部上皮幹/前驅細胞體細胞再程序化細胞不朽化
外文關鍵詞:Mouse pulmonary stem/progenitor cellssomatic reprogrammingimmortalization
相關次數:
  • 被引用被引用:0
  • 點閱點閱:215
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
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。

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.

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

Aasen, T., A. Raya, et al. (2008). "Efficient and rapid generation of induced
pluripotent stem cells from human keratinocytes." Nat Biotechnol. 26:
1276-1284.
Ambrosetti, D. C., C. Basilico, et al. (1997). "Synergistic activation of the fibroblast
growth factor 4 enhancer by sox2 and oct-3 depends on proteinprotein
interactions facilitated by a specific spatial arrangement of factor binding
sites." Mol Cell Biol. 17: 6321-6329.
Ambrosetti, D. C., H. R. Scholer, et al. (2000). "Modulation of the activity of multiple
transcriptional activation domains by the DNA binding domains mediates the
synergistic action of Sox2 and Oct-3 on the fibroblast growth factor-4
enhancer." J Biol Chem. 275: 23387-23397.
Aoi, T., K. Yae, et al. (2008). "Generation of pluripotent stem cells from adult mouse
liver and stomach cells." Science. 321: 699-702.
Armanios, M. and C. W. Greider (2005). "Telomerase and cancer stem cells." Cold
Spring Harb Symp Quant Biol. 70: 205-208.
Ben-Shushan, E., J. R. Thompson, et al. (1998). "Rex-1, a gene encoding a
transcription factor expressed in the early embryo, is regulated via Oct-3/4 and
Oct-6 binding to an octamer site and a novel protein, Rox-1, binding to an
adjacent site." Mol Cell Biol. 18 1866-1878.
Blackburn, E. H. (2001). "Switching and signaling at the telomere." Cell. 106:
661-673.
Blasco, M. A., W. Funk, et al. (1995). "Functional characterization and developmental
regulation of mouse telomerase RNA." Science. 269: 1267–1270.
Blelloch, R., Z. Wang, et al. (2006). "Reprogramming efficiency following somatic
cell nuclear transfer is influenced by the differentiation and methylation state
of the donor nucleus." Stem Cells. 24: 2007-2013.
Boeuf, H., C. Hauss, et al. (1997). "Leukemia inhibitory factor-dependent
transcriptional activation in embryonic stem cells." J Cell Biol. 138:
1207-1217.
Boyer, L. A., T. I. Lee, et al. (2005). "Core transcriptional regulatory circuitry in
human embryonic stem cells." Cell. 122: 947-956.
Boyer, L. A., D. Mathur, et al. (2006). "Molecular control of pluripotency." Curr Opin
Genet Dev. 16: 55-462.
Breuer, R., G. Zajicek, et al. (1990). "Cell kinetics of normal adult hamster bronchial
epithelium in the steady state." Am J Respir Cell Mol Biol. 2: 51-58.
Brody, J. S. and M. C. Williams (1992). "Pulmonary alveolar epithelial cell
differentiation." Annu Rev Physiol. 54: 351-371.
Burdon, T., C. Stracey, et al. (1999). "Suppression of SHP-2 and ERK signalling
promotes self-renewal of mouse embryonic stem cells." Dev Biol. 210: 30-43.
Cesare, A. J. and R. R. Reddel (2010). "Alternative lengthening of telomeres: models,
mechanisms and implications." Nat Rev Genet. 11(5): 319-330.
Chambers, I. and S. R. Tomlinson (2009). "The transcriptional foundation of
pluripotency." Development. 136: 2311-2322.
Chen, Y. C., H. S. Hsu, et al. (2008). "Oct-4 expression maintained cancer stem-like
properties in lung cancer-derived CD133-positive cells." PLoS One. 3(7):
e2637.
De Filippis, L., D. Ferrari, et al. (2008). "Immortalization of human neural stem cells
with the c-myc mutant T58A." PLoS One. 3(10): e3310.
De Filippis, L., G. Lamorte, et al. (2007). "A Novel, Immortal And Multipotent
Human Neural Stem Cell Line Generating Functional Neurons And
Oligodendrocytes." Stem Cells. 25(9): 2312-2321.
Dominguez-Sola, D., C. Y. Ying, et al. (2007). "Nontranscriptional control of DNA
replication by c-Myc." Nature. 448: 445-451.
Flores, I., A. Canela, et al. (2008). "The longest telomeres: a general signature of adult
stem cell compartments." Genes Dev. 22: 654-667.
Franken, N. A. P., H. M. Rodermond, et al. (2006). "Clonogenic assay of cells in
vitro." Nat Protoc. 1(5): 2315-2319.
Gertow, K., S. Przyborski, et al. (2007). "Isolation of human embryonic stem
cell-derived teratomas for the assessment of pluripotency." Curr Protoc Stem
Cell Biol. Chapter 1(Unit1B): 4.
Giangreco, A., S. D. Reynolds, et al. (2002). "Terminal bronchioles harbor a unique
airway stem cell population that localizes to the bronchoalveolar duct
junction." Am J Pathol. 161: 173-182.
Goldstein, S. (1990). "Replicative senescence: The human fibroblast comes of age."
Science. 249: 1129-1133.
Greenberg, R. A., R. C. Allsopp, et al. (1998). "Expression of mouse telomerase
reverse transcriptase during development, differentiation and proliferation."
Oncogene. 16: 1723-1730.
Hanna, J., S. Markoulaki, et al. (2008). "Direct reprogramming of terminally
differentiated mature B lymphocytes to pluripotency." Cell. 133: 250–264.
Harrington, L., T. McPhail, et al. (1997). "A mammalian telomerase-associated
protein." Science. 275: 973-977.
Hayashi, Y., M. Kusuda, et al. (2007). "Integrins regulate mouse embryonic stem cell
self-renewal." Stem cells. 25: 3005-3015.
Hayflick, L. and P. S. Moorhead (1961). "The serial cultivation of human diploid cell
strains." Exp Cell Res. 25: 585-621.
Hochedlinger, K. and K. Plath (2009). "Epigenetic reprogramming and induced
pluripotency." Development. 136: 509-523.
Hong, K. U., S. D. Reynolds, et al. (2001). "Clara cell secretory protein-expressing
cells of the airway neuroepithelial body microenvironment include a
label-retaining subset and are critical for epithelial renewal after progenitor
cell depletion." Am J Respir Cell Mol Biol. 24: 671-681.
Hong, K. U., S. D. Reynolds, et al. (2004). "In vivo differentiation potential of
tracheal basal cells: evidence for multipotent and unipotent subpopulations."
Am J Physiol Lung Cell Mol Physiol. 286: L643-L649.
Hooker, C. W. and P. J. Hurlin (2006). "Of Myc and Mnt." J Cell Sci. 119: 208-216.
Huangfu, D., R. Maehr, et al. (2008a). "Induction of pluripotent stem cells by defined
factors is greatly improved by small-molecule compounds." Nat Biotechnol.
26: 795–797.
Huangfu, D., K. Osafune, et al. (2008b). "Induction of pluripotent stem cells from
primary human fibroblasts with only Oct4 and Sox2." Nat Biotechnol. 26:
1269-1275.
Ichida, J. K., J. Blanchard, et al. (2009). "A small-molecule inhibitor of Tgf-β
signaling replaces Sox2 in reprogramming by inducing Nanog." Cell Stem
Cell. 5: 491-503.
Ivanova N, Dobrin R, et al. (2006). "Dissecting self-renewal in stem cells with RNA
interference." Nature. 442: 533-538.
Kim, C. B., E. L. Jackson, et al. (2005). "Identification of bronchioalveolar stem cells
in normal lung and lung cancer." Cell. 121: 823-835.
Kim, J. B., V. Sebastiano, et al. (2009). "Oct4-induced pluripotency in adult neural
stem cells." Cell. 136(3): 411-419.
Lee, C. M. and E. P. Reddy (1999). "The v-myc oncogene." Oncogene. 18(19):
2997-3003.
Lin, T., C. Chao, et al. (2005). "p53 induces differentiation of mouse embryonic stem
cells by suppressing Nanog expression." Nat Cell Biol. 7: 165-171.
Ling, T. Y., M. D. Kuo, et al. (2006). "Identification of pulmonary Oct-4+
stem/progenitor cells and demonstration of their susceptibility to SARS
coronavirus (SARS-CoV) infection in vitro." Proc Natl Acad Sci U S A.
103(25): 9530-9535.
Lingner, J., T. R. Hughes, et al. (1997). "Reverse transcriptase motifs in the catalytic
subunit of telomerase." Science. 276: 561-567.
Liu, L., S. N. Saldanha, et al. (2004). "Epigenetic regulation of human telomerase
reverse transcriptase promoter activity during cellular differentiation." Genes
Chromosomes Cancer. 41: 26-37.
Maherali, N. and K. Hochedlinger (2008). "Guidelines and techniques for the
generation of induced pluripotent stem cells." Cell Stem Cell. 3: 595-605.
Maherali, N., R. Sridhara, et al. (2007 ). "Directly reprogrammed fibroblasts show
global epigenetic remodeling and widespread tissue contribution." Cell Stem
Cell. 1: 55-70.
Marion, R. M., K. Strati, et al. (2009). "Telomeres acquire embryonic stem cell
characteristics in induced pluripotent stem cells." Cell Stem Cell. 4: 141-154.
Marson, A., S. S. Levine, et al. (2008). "Connecting microRNA genes to the core
transcriptional regulatory circuitry of embryonic stem cells." Cell. 134:
521-533.
Martinez-Serrano, A., F. J. Rubio, et al. (2001 ). "Human neural stem and progenitor
cells: in vitro and in vivo properties, and potential for gene therapy and cell
replacement in the CNS." Curr Gene Ther. 1(3): 279-299.
Mason, R. J. and M. C. Williams (1977). "Type II alveolar cell. Defender of the
alveolus." Am Rev Respir Dis. 115(6 Pt 2): 81-91.
Mathew, R., W. Jia, et al. (2010). "Robust activation of the human but not mouse
telomerase gene during the induction of pluripotency." FASEB J. 24: 1-14.
Matsuda, T., T. Nakamura, et al. (1999). "STAT3 activation is sufficient to maintain
an undifferentiated state of mouse embryonic stem cells." EMBO J. 18(15):
4261-4269.
Meyer, N., S. S. Kim, et al. (2006). "The Oscar-worthy role of Myc in apoptosis. ."
Semin Cancer Biol. 16: 275-287.
Meyerson, M., C. M. Counter, et al. (1997). "hEST2, the putative human telomerase
catalytic subunit gene, is up-regulated in tumor cells and during
immortalization." Cell. 90: 785-795.
Mikkelsen, T. S., J. Hanna, et al. (2008). "Dissecting direct reprogramming through
integrative genomic analysis." Nature. 454: 49-55.
Nakagawa, M., M. Koyanagi, et al. (2007). "Generation of induced pluripotent stem
cells without Myc from mouse and human fibroblasts." Nat Biotechnol. 26:
101-106.
Nishimoto, M., Fukushima, et al. (1999). "The gene for the embryonic stem cell
coactivator UTF1 carries a regulatory element which selectively interacts with
a complex composed of Oct-3/4 and Sox-2." Mol Cell Biol. 19: 5453-5465.
Niwa, H. (2007). "How is pluripotency determined and maintained?." Development.
134: 635-646.
Niwa, H., T. Burdon, et al. (1998). "Self-renewal of pluripotent embryonic stem cells
is mediated via activation of STAT3." Genes Dev. 12: 2048 -2060.
Okita, K., T. Ichisaka, et al. (2007). "Generation of germline-competent induced pluripotent stem cells." Nature. 448: 313-317.
Okumura-Nakanishi, S., M. Saito, et al. (2005). "Oct-3/4 and Sox2 regulate Oct-3/4
gene in embryonic stem cells." J Biol Chem. 280: 5307- 5317.
Ouellette, M. M., M. Liao, et al. (2000). "Subsenescent telomere lengths in fibroblasts
immortalized by limiting amounts of telomerase." J Biol Chem. 275(14):
10072-10076.
Paling, N. R., H. Wheadon, et al. (2004). "Regulation of embryonic stem cell
self-renewal by phosphoinositide 3-kinase-dependent signaling." J Biol Chem.
279: 48063- 48070.
Peake, J. L., S. D. Reynolds, et al. ( 2000). "Alteration of pulmonary neuroendocrine
cells during epithelial repair of naphthalene-induced airway injury." Am J
Pathol. 156: 279-286.
Rock, R. J., M. W. Onaitis, et al. (2009). "Basal cells as stem cells of the mouse
trachea and human airway epithelium." Proc Natl Acad Sci U S A.. 106 (31):
12771-12775.
Ryder, E. F., E. Y. Snyder, et al. (1990). "Establishment and characterization of
multipotent neural cell lines using retrovirus vector-mediated oncogene
transfer." J Neurobiol. 21(2): 356-375.
Shi, Y., J. T. Do, et al. (2008). "A combined chemical and genetic approach for the
generation of induced pluripotent stem cells." Cell Stem Cell. 2: 525-528.
Shippen-Lentz, D. and E. H. Blackburn (1990). "Functional evidence for an RNA
template in telomerase." Science. 247: 546-552.
Stadtfeld, M., K. Brennand, et al. (2008). "Reprogramming of pancreatic β cells into
induced pluripotent stem cells." Current Biology. 18: 890-894.
Sun, N., N. J. Panetta, et al. (2009 ). "Feeder-free derivation of induced pluripotent
stem cells from adult human adipose stem cells." Proc Natl Acad Sci U S A.
106 (37): 15720–15725
Takahashi, K. and S. Yamanaka (2006). "Induction of pluripotent stem cells from
mouse embryonic and adult fibroblast cultures by defined factors." Cell.
126( 663-676).
Weinberg, R. A. (1998). "Telomeres: Bumps on the road to immortality." Nature.
396(6706): 23-24.
Wernig, M., A. Meissner, et al. (2008). "C-Myc is dispensable for direct
reprogramming of mouse fibroblasts." Cell Stem Cell. 2: 10-12.
Wikenheiser, K. A., D. K. Vorbroker, et al. (1993). "Production of immortalization
distal respiratory epithelial cell lines from surfactant protein C/simian virus 40
large tumor antigen transgenic mice." Proc Natl Acad Sci U S A. 90:
11029-11033.
Williams, R. L., D. J. Hilton, et al. (1988). "Myeloid leukaemia inhibitory factor
maintains the developmental potential of embryonic stem cells." Nature. 336:
684-687.
Wright, W. E., O. M. Pereira-Smith, et al. (1989). "Reversible cellular senescence:
implications for immortalization of normal human diploid fibroblasts." Mol
Cell Biol. 9(7): 3088-3092.
Yeh, E., M. Cunningham, et al. (2004). "A signalling pathway controlling c-Myc
degradation that impacts oncogenic transformation of human cells." Nat Cell
Biol. 6(4): 308-318.
Yu, J., M. A. Vodyanik, et al. (2007). "Induced pluripotent stem cell lines derived
from human somatic cells." Science. 318(5858): 1917-1920.
Yuan, H., N. Corbi, et al. (1995). "Developmental-specific activity of the FGF-4
enhancer requires the synergistic action of Sox2 and Oct-3." Genes Dev. 9
2635-2645.
Yuan, P., H. Kadara, et al. (2010). "Sex determining region Y-box 2 (SOX2) is a
potential cell-lineage gene highly expressed in the pathogenesis of squamous
cell carcinomas of the lung." PLoS One. 5(2): e9112.

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔