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研究生:姚道禮
研究生(外文):Tu-Lai Yew
論文名稱:p21基因剔除對人類骨髓間葉幹細胞之增殖能力、細胞幹性及骨分化潛力之影響
論文名稱(外文):Ex vivo targeting of p21Cip1/Waf1 enhances proliferation, the expression of stemness markers and osteogenic potential of human bone marrow-derived mesenchymal stem cells
指導教授:陳恆理陳恆理引用關係洪士杰
指導教授(外文):Hen-Li ChenShih-Chieh Hung
學位類別:博士
校院名稱:國立陽明大學
系所名稱:口腔生物研究所
學門:醫藥衛生學門
學類:牙醫學類
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:98
中文關鍵詞:骨髓間葉幹細胞老化p21骨分化潛力骨癒合
外文關鍵詞:mesenchymal stem cellssenescencep21osteogenesisbone repair
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中文摘要
使用骨髓間葉幹細胞(mesenchymal stem cells)的細胞性治療在骨再生治療上深具潛力。臨床應用時常須在體外大量增殖骨髓間葉幹細胞,以獲得足夠數量的細胞。體外培養會造成骨髓間葉幹細胞老化,使細胞增生變慢,失去幹細胞特性,並降低治療效果,但目前仍無有效且安全的方法來解決幹細胞老化的問題。過去研究顯示細胞週期調控因子p21可能跟細胞老化有關,我們認為p21在骨髓間葉幹細胞老化的過程中可能扮演重要角色。本研究目的在探討p21在人類骨髓間葉幹細胞老化中的角色。本研究結果顯示骨髓間葉幹細胞隨著體外增殖培養,有p21上升和老化的現象。利用慢病毒基因載體系統(lentiviral system) 短髮夾核醣核酸(shRNA)方式降低已老化間葉幹細胞的p21表達,發現有助於提高體外的細胞增殖速率、細胞幹性標記及骨生成潛能。更重要的是已老化間葉幹細胞的p21降低後,在小鼠顱頂骨缺損模型研究顯示可加強其骨癒合能力。p21表達降低的骨髓間葉幹細胞具較強端粒酶(telomerase)活性和較長端粒(telomere),但仍有正常完整的染色體核型,且未發現有致癌性。本研究證實p21在人類骨髓間葉幹細胞的老化的過程中佔重要角色。在預防或減少間葉幹細胞在體外培養過程中所產生的老化現象方面,降低p21可能成為一個有效且安全的策略。
Abstract
Cell-based therapies using bone marrow-derived mesenchymal stem cells (MSCs) demonstrate great potential in bone regenerative therapies. Ex vivo expansion of MSCs is often required to generate adequate cell numbers in clinical applications. Senescence of MSCs occurs along with ex vivo passages and results in lower proliferation rate, loss of stemness and compromised therapeutic potential. However, currently no effective and safe method is available to solve the senescence problem. Previous studies indicated that a cell cycle regulator, p21, may be associated with cell senescence. We hypothesized that p21 may play an important role in the senescence of bone marrow-derived MSCs. The purpose of this research was to determine the role of p21 expression in the senescence of human bone marrow-derived MSCs. The results indicated that MSCs increased in p21 expression and became senescent along with ex vivo expansion. Lentiviral transduction of senescent MSCs with p21 shRNAs was able to increase their proliferation capacity, expression of stemness markers, and osteogenic potential in vitro. More importantly, the reduction of p21 expression enhanced the bone repair capacity of senescent MSCs in a mouse calvarial defect model. The p21-knockdowned MSCs showed increased telomerase activity and telomere length but maintained normal chromosome integrity and did not acquire tumorigenic potential. In conclusion, p21 plays an important role in senescence of human bone marrow-derived MSCs. The knockdown of p21 may become an effective and safe strategy to prevent or reduce the senescence of MSCs during ex vivo expansion.
CONTENTS
TABLE OF CONTENT 1
LIST OF ABBREVIATIONS 8
中文摘要 11
ENGLISH ABSTRACT 13
INTRODUCTION 15
STUDY PURPOSE 20
SPECIFIC AIMS 21
AIM 1: TO ESTABLISH AND VERIFY THE SENESCENT MSCS 21
AIM 2: TO ESTABLISH P21 KNOCKDOWN MSCS AND DETERMINE THE EFFECT OF P21 KNOCKDOWN ON SENESCENT MSCS IN VITRO 21
AIM 3: TO DETERMINE THE EFFECT OF P21 KNOCKDOWN ON SENESCENT MSCS IN VIVO. 22
AIM 4: TO INVESTIGATE THE TUMORIGENIC POTENTIAL OF TRANSDUCED MSCS. 23
MATERIALS AND METHODS 24
ISOLATION AND CULTURES OF HUMAN MSCS 24
CELL GROWTH ASSAY 25
CELL CYCLE ANALYSIS 26
BROMODEOXYURIDINE (BRDU) INCORPORATION ASSAY 27
IN VITRO DIFFERENTIATION 28
ALKALINE PHOSPHATASE ACTIVITY 28
LENTIVIRAL-MEDIATED RNAI 29
WESTERN BLOTTING 30
REAL-TIME PCR 31
SCAFFOLD PREPARATION FOR IN VIVO EXPERIMENTS 32
SCANNING ELECTRON MICROSCOPY 33
CALVARIAL DEFECT ANIMAL MODEL 34
RADIOGRAPHY, MICRO-CT AND DUAL ENERGY X-RAY ABSORPTIOMETRY (DEXA) ANALYSIS 35
HISTOLOGY 36
TELOMERE LENGTH ANALYSIS AND TELOMERE REPEAT AMPLIFICATION PROTOCOL (TRAP). 37
COMPARATIVE GENOMIC HYBRIDIZATION (CGH) 38
TUMORIGENICITY ASSAY 38
STATISTICAL ANALYSIS 39
RESULTS 40
MSCS DECREASE IN THE CELL PROLIFERATION RATE AND INCREASE IN SENESCENCE MARKERS FOLLOWING EXPANSION 40
DECREASED EXPRESSION OF STEMNESS MARKERS AND REDUCED OSTEOGENIC POTENTIAL IN LATE-PASSAGE MSCS 42
KNOCKDOWN OF P21 IN LATE-PASSAGE MSCS INHIBITS SENESCENCE AND INCREASES CELL PROLIFERATION, EXPRESSION OF STEMNESS MARKERS, AND OSTEOGENIC POTENTIAL 43
THE ARCHITECTURE OF COLLAGEN SPONGE 45
KNOCKDOWN OF P21 IN LATE-PASSAGE MSCS INCREASES BONE HEALING CAPACITY WHEN TRANSPLANTED IN MICE CALVARIAL DEFECTS 45
NEW BONE FORMATION IS MAINLY FROM TRANSPLANTED MSCS. 46
KNOCKDOWN OF P21 IN LATE-PASSAGE MSCS INCREASES TELOMERASE ACTIVITY AND TELOMERE LENGTH AND DID NOT INDUCE TRANSFORMATION 48
DISCUSSION 50
REFERENCES 55
TABLE 66
TABLE 1. PRIMER SETS FOR REAL-TIME PCR 66
FIGURE LEGENDS 67
FIGURE 1. LATE-PASSAGE MSCS DECREASE IN CELL GROWTH 67
FIGURE 2. LATE-PASSAGE MSCS DECREASE IN CELL NUMBER BUT NOT INCREASE IN APOPTOSIS 68
FIGURE 3. P21 IS THE MAIN CELL CYCLE REGULATOR INCREASED IN LATE-PASSAGE MSCS 69
FIGURE 4. LATE-PASSAGE MSCS INCREASE IN SENESCENCE 70
FIGURE 5. LATE-PASSAGE MSCS DECREASE IN THE EXPRESSION OF EMBRYONIC MARKERS BY QUANTITATIVE RT-PCR 71
FIGURE 6. LATE-PASSAGE MSCS DECREASE IN THE EXPRESSION OF EMBRYONIC MARKERS BY WESTERN BLOT 72
FIGURE 7. LATE-PASSAGE MSCS DECREASE IN THE DIFFERENTIATION GENE PROFILE 73
FIGURE 8. LATE-PASSAGE MSCS DECREASE THE OSTEOGENIC POTENTIAL IN VITRO 74
FIGURE 9. LATE-PASSAGE MSCS DECREASE THE MINERALIZATION POTENTIAL IN VITRO 75
FIGURE 10. KNOCKDOWN OF P21 IN LATE-PASSAGE MSCS INHIBITS SENESCENCE 77
FIGURE 11. KNOCKDOWN OF P21 IN LATE-PASSAGE MSCS STIMULATES PROLIFERATION 78
FIGURE 12. KNOCKDOWN OF P21 IN LATE-PASSAGE MSCS INCREASES IN CELL NUMBER BUT NOT INCREASES APOPTOSIS IN SCRAMBLE CELLS 79
FIGURE 13. KNOCKDOWN OF P21 IN LATE-PASSAGE MSCS INCREASES IN THE EXPRESSION OF EMBRYONIC MARKERS BY QUANTITATIVE RT-PCR 80
FIGURE 14. KNOCKDOWN OF P21 IN LATE-PASSAGE MSCS INCREASES IN THE EXPRESSION OF EMBRYONIC MARKERS BY WESTERN BLOT 81
FIGURE 15. KNOCKDOWN OF P21 IN LATE-PASSAGE MSCS INCREASES IN THE DIFFERENTIATION GENE PROFILE 82
FIGURE 16. KNOCKDOWN OF P21 IN LATE-PASSAGE MSCS INCREASES THE OSTEOGENIC POTENTIAL IN VITRO 83
FIGURE 17. KNOCKDOWN OF P21 IN LATE-PASSAGE MSCS INCREASES THE MINERALIZATION POTENTIAL IN VITRO 84
FIGURE 18. THE ARCHITECTURE OF COLLAGEN SPONGE 86
FIGURE 19. THE QUANTIFICATION OF BONE FORMATION REVEALED BY RADIOGRAPHIC ANALYSIS 87
FIGURE 20. KNOCKDOWN OF P21 IN LATE-PASSAGE MSCS INCREASE BONE VOLUME 89
FIGURE 21. KNOCKDOWN OF P21 IN LATE-PASSAGE MSCS INCREASE IN VIVO BONE REPAIRING ABILITY 91
FIGURE 22. EVALUATION OF BONE REGENERATION BY HISTOLOGICAL ANALYSIS 92
FIGURE 23. EVALUATION OF BONE REGENERATION BY HISTOLOGICAL ANALYSIS 94
FIGURE 24. KNOCKDOWN OF P21 IN LATE-PASSAGE MSCS INCREASES TELOMERASE ACTIVITY AND TELOMERE LENGTH 95
FIGURE 25. KNOCKDOWN OF P21 IN LATE-PASSAGE MSCS SHOWED NORMAL COMPARATIVE GENOMIC HYBRIDIZATION 96
FIGURE 26. KNOCKDOWN OF P21 IN LATE-PASSAGE MSCS DID NOT INDUCE TUMORIGENIC TRANSFORMATION IN VITRO 97
FIGURE 27. KNOCKDOWN OF P21 IN LATE-PASSAGE MSCS DID NOT INDUCE TUMORIGENIC TRANSFORMATION IN VIVO 98


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