跳到主要內容

臺灣博碩士論文加值系統

(44.192.67.10) 您好!臺灣時間:2024/11/14 22:53
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:陳立欣
研究生(外文):Li-Hsin Chen
論文名稱:PGC-1α於人類骨髓幹細胞的過度表現:粒線體之調控與生長速率之抑制
論文名稱(外文):Overexpression of PGC-1α in Human Bone Marrow Stem Cells: the Regulation of Mitochondria and the Inhibition of Growth Rate
指導教授:古宏海
指導教授(外文):Hung-Hai Ku
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:解剖暨細胞生物學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:84
中文關鍵詞:幹細胞老化
外文關鍵詞:stem cellsPGC-1αsenescence
相關次數:
  • 被引用被引用:0
  • 點閱點閱:321
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
幹細胞具有一種獨特的能力,稱為自我更新(self-renewal),能夠讓幹細胞在生物體內不斷分裂,且同時保有多能(pluripotency)分化的能力,而不至於隨著生物體老化而喪失。另一方面,粒線體已經被發現參與調控細胞的生命期與細胞老化;而人類胚胎幹細胞中,粒線體較小,且數量較少;細胞核內的PGC-1α屬於一個輔助活化因子(coactivator)的家族,在許多種細胞中,能夠調節能量代謝並增加粒線體質量。為了探討幹細胞是否會因為細胞內粒線體質量增加,而改變原先保持靜止(quiescent)的狀態,故先分離人類骨髓幹細胞,經由腺病毒表現系統將PGC-1α送入細胞。發現在人類骨髓幹細胞中,過度表現PGC-1α確實能夠增加粒線體質量到二倍以上。與控制組相比較,細胞送入PGC-1α後,Doubling time延長到三倍時間。而在送入PGC-1α第七天的人類骨髓幹細胞,表現senescence-associated β-galactosidase (SA-β-Gal)活性占全體細胞的40%,而控制組僅7%。再者,reactive oxygen species(ROS)產量在過度表現PGC-1α的細胞中亦上升達2.5倍。因此,研究結果顯示,藉由腺病毒送入PGC-1α能夠增加骨髓幹細胞內的粒線體質量,並造成細胞生長速率下降,更可引發老化反應;即粒線體在維持幹細胞的生命期上,的確扮演了重要的角色。
Stem cells have a unique “self-renewal” ability. This enables them to continuously divide throughout the life of the organism and to maintain their pluripotency without aging. On the other hand, it has been reported that mitochondrion plays an important role in regulating cellular life span and cellular senescence. Mitochondria are few and small in human embryonic stem cells. In addition, PGC-1α belongs to a family of coactivator. It has been proved to regulate energy metabolism and increase mitochondrial mass in many cell types. In order to investigate if stem cells would alter their quiescent status by increasing mitochondrial mass, human bone marrow stem cells (BMSCs) were isolated and PGC-1α was overexpressed in these cells through adenoviral expression system. It was found that overexpression of PGC-1α do increase mitochondrial mass by two times in BMSCs. As compared to the control, the doubling time increased 3 folds after BMSCs were infected by PGC-1α incorporated adenovirus. The percentages of expressing senescence-associated β-galactosidase (SA-β-Gal) activity were significantly increased to 40% in Day 7 overexpressed PGC-1α BMSCs as compared to 7% in control. Moreover, ROS production in overexpressed PGC-1α BMSCs was doubled as compared to those in BMSCs infected by vector only. In conclusion, these results suggested that by increasing mitochondrial mass via adenoviral-mediated expression of PGC-1α, a reduction in cell growth and cellular senescence could be induced in BMSCs. Therefore, mitochondria play a critical role in regulating cellular life span.
目 錄………………………………………………………… 2
誌 謝………………………………………………………… 4
中文摘要…………………………………………………… 5
英文摘要…………………………………………………… 6
英文縮寫表………………………………………………… 7
圖表目次…………………………………………………… 9
第一章 緒論……………………………………………… 10
(一) 幹細胞之簡介 11
(二) 老化之簡介 13
(三) 幹細胞與老化的關係 15
(四) 粒線體與老化的關係 16
(五) PGC-1α對能量代謝與粒線體的影響 18
(六) 目標與實驗設計 20
第二章 材料與方法…………………………………………………… 23
(一) 骨髓幹細胞(Bone marrow stem cell;BMSC)的分離與分化
24
(二) RNA的萃取 26
(三) RT-PCR 27
(四) 基因送入系統 27
(五) 腺病毒表現系統(Adenovirus expression system) 29
(六) 微陣列分析(Microarray analysis) 29
(七) 西方點墨法 31
(八) 轉染質體pDsRed-Mt,並以共軛焦顯微鏡觀察 31
(九) 氫化乙啡啶染色(Hydroethidine staining) 31
(十) 粒線體質量(Mitochondrial mass)測定 32
(十一) Senescence associated β-galactosidase staining 32
(十二) 統計分析 32
第三章 結果…………………………………………………… 33
(一) 骨髓幹細胞的分離 34
(二) 骨髓幹細胞之分化能力 34
(三) 不同方法送入基因至骨髓幹細胞效率之評估 35
(四) 骨髓幹細胞過度表現PGC-1α對調控粒線體的影響 37
(五) 骨髓幹細胞過度表現PGC-1α對生命期(life span)造成的影響
38
(六) 骨髓幹細胞過度表現PGC-1α之微陣列基因結果分析
40
第四章 討論……………………………………………… 42
參考文獻…………………………………………………… 50
附表及圖…………………………………………………… 58
Agarwal S., Sohal R.S. (1994) Aging and protein oxidative damage. Mech. Ageing Dev. 75: 11–19.
Banfi A., Muraglia A., Dozin B., Mastrogiacomo M., Cancedda R., Quarto R. (2000) Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: implications for their use in cell therapy. Exp. Hematol. 28: 707–715.
Baxter M.A., Wynn R.F., Jowitt S.N., Wraith J.E., Fairbairn L.J., Bellantuono I. (2004) Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion. Stem Cells 22: 675–682.
Bertoni-Freddari C., Fattoretti P., Casoli T., Spagna C., Meier-Ruge W. (1994) Morphological alterations of synaptic mitochondria during aging. Ann NY Acad Sci 717: 137–149.
Bonab M.M., Alimoghaddam K., Talebian F., Ghaffari S.H., Ghavamzadeh A., Nikbin B. (2006) Aging of mesenchymal stem cell in vitro. BMC Cell Biol. 7: 14.
Brooks A.R., Harkins R.N., Wang P., Qian H.S., Liu P., Rubanyi G.M. (2004) Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle. J. Gene Med. 4: 395–404.
Bruder S.P., Jaiswal N., Haynesworth S.E. (1997) Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J. Cell Biochem. 64: 278–294.
Butow R.A., Bahassi E.M. (1999) Adaptive thermogenesis: orchestrating mitochondrial biogenesis. Curr. Biol. 9: R767–R769.
Chen Q., Fischer A., Reagan J.D., Yan L.J., Ames B.N. (1995) Oxidative DNA damage and senescence of human diploid fibroblast cells. Proc. Natl Acad. Sci. USA 92: 4337–4341.
Cournil A., Kirkwood T.B.L. (2001) If you would live long, choose your parents well. Trends Genet. 17: 233–235.
De Ugarte D.A., Alfonso Z., Zuk P.A., Elbarbary A., Zhu M., Ashjian P., Benhaim P., Hedrick M.H., Fraser J.K. (2003) Differential expression of stem cell mobilization-associated molecules on multi-lineage cells from adipose tissue and bone marrow. Immunol. Lett. 89: 267–270.
Deng W., Obrocka M., Fischer I., Prockop D.J. (2001) In vitro differentiation of human marrow stromal cells into early progenitors of neural cells by conditions that increase intracellular cyclic AMP. Biochem. Biophys. Res. Commun. 282: 148–152.
Dimri G.P., Lee X., Basile G., Acosta M., Scott G., Roskelley C., Medrano E.E., Linskens M., Rubelj I., Pereira-Smith O. Peacocke M., Campisi J. (1995) A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl Acad. Sci. USA 92: 9363–9367.
Dimri G.P., Testori A., Acosta M., Campisi J. (1996) Replicative senescence, aging and growth-regulatory transcription factors. Biol. Signals 5: 154–162.
Esterbauer H., Oberkofler H., Krempler F., Patsch W. (1999) Human peroxisome proliferator activated receptor gamma coactivator 1 (PPARGC1) gene: cDNA sequence, genomic organization, chromosomal localization, and tissue expression. Genomics 62: 98–102.
Finch C.E., Kirkwood T.B.L. (2000) Chance, Development and Aging (New York: Oxford University Press).
Fridovich, I. (2004) Mitochondria: are they the seat of senescence? Aging Cell 3: 13–16.
Goto M., Terada S., Kato M., Katoh M., Yokozeki T., Tabata I., Shimokawa T. (2000) cDNA cloning and mRNA analysis of PGC-1 in epitrochlearis muscle in swimming-exercised rats. Biochem. Biophys. Res. Commun. 274: 350–354.
Halliwell B., Gutteridge J.M. (1999) Free Radicals in Biology and Medicine, 3rd ed. Oxford University Press, Oxford.
He, T.C., Zhou S., da Costa L.T., Yu J., Kinzler K.W., Vogelstein B. (1998) A simplified system for generating recombinant adenoviruses. Proc. Natl Acad. Sci. USA 95: 2509-2514.
Herndon L.A., Schmeissner P.J., Dudaronek J.M., Brown P.A., Listner K.M., Sakano Y., Paupard M.C., Hall D.H., and Driscol M. (2002). Stochastic and genetic factors influence tissue-specific decline in ageing C. elegans. Nature 419: 1117–1123.
Herzberg N.H., Zwart R., Wolterman R.A., Ruiter J.P., Wanders R.J., Bolhuis P.A., van den Bogert C. (1993) Differentiation and proliferation of respiration-deficient human myoblasts. Biochim Biophys Acta. 1181:63-67.
Jaiswal N., Haynesworth S.E., Caplan A.I., Bruder S.P. (1997) Osteogenic differentiation of purified, culture expanded human mesenchymal stem cells in vitro. J. Cell. Biochem. 64: 295–312.
Jiang Y., Vaessen B., Lenvik T., Blackstad M., Reyes M., Verfaillie C.M. (2002) Multipotent progenitor cells can be isolated from postnatal murine bone marrow, muscle, and brain. Exp. Hematol. 30: 896–904.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top