跳到主要內容

臺灣博碩士論文加值系統

(44.220.44.148) 您好!臺灣時間:2024/06/21 17:03
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:黃君豪
研究生(外文):Huang, Chun-Hsiang
論文名稱:探討p21與ZAC1在人類脂源性間葉幹細胞分化成 脂肪及軟骨細胞的角色
論文名稱(外文):The study of the functional roles of p21 and ZAC1 in the differentiation of human adipose-derived stromal cell into adipocyte and chondrocyte
指導教授:黃世明黃世明引用關係
指導教授(外文):Huang, Shih-Ming
口試委員:詹益新王偉銘戴建國黃紀榕
口試委員(外文):James, Yi-Hsin ChanWang, Wei-mingTai, Chien-KuoHuang, Chi-Jung
口試日期:2011-06-01
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:60
中文關鍵詞:脂肪幹細胞分化
外文關鍵詞:adipose-derived stem celldifferentiationp21ZAC1
相關次數:
  • 被引用被引用:0
  • 點閱點閱:242
  • 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
人類脂源性間葉幹細胞(Human adipose-derived stromal cells)是一種具有多重分化潛力的幹細胞;可以走向脂肪、軟骨、硬骨等不同的分化。在組織醫學工程中,具有高度的應用的潛力,未來可望廣泛用於治療疾病。而在人類脂源性間葉幹細胞分化的過程中,發現細胞週期抑制蛋白p21會隨者不同的分化路徑,有截然不同的表現量。已知細胞周期抑制蛋白p21,可以調控細胞的分化能力以及決定分化的程度;本實驗室的研究顯示轉錄因子ZAC1會結合在p21的啟動子上,進而調控p21的基因與蛋白質表現量。因此本論文將探討在人類脂源性間葉幹細胞分化的過程中,p21扮演的功能及轉錄因子ZAC1是否透過影響p21的路徑決定分化的走向。首先經由流式細胞儀分析萃取細胞的細胞表面抗原,顯示符合間葉幹細胞的特性;萃取的細胞進行不同的分化誘導,經染色也證實具有多元分化潛力的能力。在分化成脂肪細胞與軟骨細胞的過程中,發現到p21會隨著分化而有所增減;進一步將萃取的細胞內的ZAC1或p21剔除時,經由染色、定量PCR與西方點墨法分析,當剔除細胞的ZAC1或p21基因,會導致細胞分化成脂肪與軟骨程度有所增減。結果顯示ZAC1或p21基因似乎會影響人類脂源間葉幹細胞分化的過程。推測轉錄因子ZAC1可能藉由調控特定基因的表現,其中包括一些參與脂肪與軟骨細胞分化的重要基因;而p21的表現,則有助於細胞停留在S期,使細胞減少增生進而轉向分化的途徑。
Human adipose-derived stromal cells (hASCs) have multi-differentiation potentials by differentiating cells with adipogenic, chondrogenic, osteogenic. In clinical cell-based therapy and tissue engineering, hASCs have highly potential applications. In the future, hASCs are expected to widely use for various clinical therapies. In the process of differentiation, p21, a well-known cyclin-dependent kinase inhibitor, variedly expresses in various differentiations to determine the degree of differentiation. Our previous study demonstrated that transcription factor ZAC1 can regulate p21 gene and protein expression through the direct binding in p21 promoter. In this study, we tried to examine the role of p21 in the hASCs differentiation and whether ZAC1 might determine the direction of differentiation through the p21-dependent mechanism. First, the flow cytometry analysis demonstrated that extracted cells expressed high levels of various stem cell-related antigens and had the ability to differentiate into various cell types using the staining method. During adipogenesis and chondrogenesis, the expression of p21 depended on the differentiation situations. Using the staining, RT-PCR, and Western blot analysis is to examine the effect of knockdown ZAC1 or p21 expression in hASC, suggesting that ZAC1 and p21 play the role in the hASC differentiations. Summary, ZAC1 may regulate a set of specific genes for adipogenesis and chondrogenesis; p21 could keep cells in S phase to reduce cell proliferation and turn into differentiation procedure.
目錄 I
圖目錄 III
縮寫表 V
中文摘要 VI
英文摘要 VII
第一章 緒論 1
第二章 實驗材料與方法 6
第一節 實驗材料 6
第二節 實驗方法 10
第三章 結果 20
第一節 建立從脂肪組織中萃取脂源性間葉幹細胞的模式 20
第二節 誘導脂源性間葉幹細胞分化成脂肪細胞 21
第三節 誘導脂源性間葉幹細胞分化成軟骨細胞 21
第四節 利用慢病毒(lentivirus)系統降低脂源性間葉幹細胞株中ZAC1及p21的表現量 22
第五節 降低脂源性間葉幹細胞株中ZAC1及p21的表現量對於誘導脂肪分化的影響 24
第六節 降低脂源性間葉幹細胞株中ZAC1及p21的表現量對於誘導軟骨分化的影響 24
第四章 討論 26
第一節 建立從脂肪組織中萃取脂源性間葉幹細胞之模式 26
第二節 p21在幹細胞的脂肪分化中可能扮演的角色 27
第三節 ZAC1在脂肪分化中可能扮演的角色 29
第四節 p21在軟骨分化中可能扮演的角色 30
第五節 ZAC1在軟骨分化中可能的扮演角色 31
圖............... 33
參考文獻. 47

1. Abbas, T., and Dutta, A. (2009). p21 in cancer: intricate networks and multiple activities. Nat Rev Cancer 9, 400-414.
2.Akiyama, H., Chaboissier, M. C., Martin, J. F., Schedl, A., and de Crombrugghe, B. (2002). The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev 16, 2813-2828.
3.Banerjee, S. S., Feinberg, M. W., Watanabe, M., Gray, S., Haspel, R. L., Denkinger, D. J., Kawahara, R., Hauner, H., and Jain, M. K. (2003). The Kruppel-like factor KLF2 inhibits peroxisome proliferator-activated receptor-gamma expression and adipogenesis. J Biol Chem 278, 2581-2584.
4.Barrilleaux, B., Phinney, D. G., Prockop, D. J., and O'Connor, K. C. (2006). Review: ex vivo engineering of living tissues with adult stem cells. Tissue Eng 12, 3007-3019.
5.Barz, T., Hoffmann, A., Panhuysen, M., and Spengler, D. (2006). Peroxisome proliferator-activated receptor gamma is a Zac target gene mediating Zac antiproliferation. Cancer Res 66, 11975-11982.
6.Bi, W., Huang, W., Whitworth, D. J., Deng, J. M., Zhang, Z., Behringer, R. R., and de Crombrugghe, B. (2001). Haploinsufficiency of Sox9 results in defective cartilage primordia and premature skeletal mineralization. Proc Natl Acad Sci U S A 98, 6698-6703.
7.Bobick, B. E., Chen, F. H., Le, A. M., and Tuan, R. S. (2009). Regulation of the chondrogenic phenotype in culture. Birth Defects Res C Embryo Today 87, 351-371.
8.Coppock, H. A., Gilham, D. E., Howell, A., and Clarke, R. B. (2007). Cyclin-dependent kinase inhibitors and basement membrane interact to regulate breast epithelial cell differentiation and acinar morphogenesis. Cell Prolif 40, 721-740.
9.Coqueret, O., and Gascan, H. (2000). Functional interaction of STAT3 transcription factor with the cell cycle inhibitor p21WAF1/CIP1/SDI1. J Biol Chem 275, 18794-18800.
10.Devgan, V., Mammucari, C., Millar, S. E., Brisken, C., and Dotto, G. P. (2005). p21WAF1/Cip1 is a negative transcriptional regulator of Wnt4 expression downstream of Notch1 activation. Genes Dev 19, 1485-1495.
11.Djouad, F., Bouffi, C., Ghannam, S., Noel, D., and Jorgensen, C. (2009). Mesenchymal stem cells: innovative therapeutic tools for rheumatic diseases. Nat Rev Rheumatol 5, 392-399.
12.Dotto, G. P. (2000). p21(WAF1/Cip1): more than a break to the cell cycle? Biochim Biophys Acta 1471, M43-56.
13.Estes, B. T., Diekman, B. O., Gimble, J. M., and Guilak, F. (2010). Isolation of adipose-derived stem cells and their induction to a chondrogenic phenotype. Nat Protoc 5, 1294-1311.
14.Grassel, S., and Ahmed, N. (2007). Influence of cellular microenvironment and paracrine signals on chondrogenic differentiation. Front Biosci 12, 4946-4956.
15.Gray, S., Feinberg, M. W., Hull, S., Kuo, C. T., Watanabe, M., Sen-Banerjee, S., DePina, A., Haspel, R., and Jain, M. K. (2002). The Kruppel-like factor KLF15 regulates the insulin-sensitive glucose transporter GLUT4. J Biol Chem 277, 34322-34328.
16.Grigoriadis, A. E., Heersche, J. N., and Aubin, J. E. (1988). Differentiation of muscle, fat, cartilage, and bone from progenitor cells present in a bone-derived clonal cell population: effect of dexamethasone. J Cell Biol 106, 2139-2151.
17.Hong, J., Samudio, I., Liu, S., Abdelrahim, M., and Safe, S. (2004). Peroxisome proliferator-activated receptor gamma-dependent activation of p21 in Panc-28 pancreatic cancer cells involves Sp1 and Sp4 proteins. Endocrinology 145, 5774-5785.
18.Huang, S. M., Huang, S. P., Wang, S. L., and Liu, P. Y. (2007). Importin alpha1 is involved in the nuclear localization of ZAC1 and the induction of p21WAF1/CIP1 by ZAC1. Biochem J 402, 359-366.
19.Inoue, N., Yahagi, N., Yamamoto, T., Ishikawa, M., Watanabe, K., Matsuzaka, T., Nakagawa, Y., Takeuchi, Y., Kobayashi, K., Takahashi, A., et al. (2008). Cyclin-dependent kinase inhibitor, p21WAF1/CIP1, is involved in adipocyte differentiation and hypertrophy, linking to obesity, and insulin resistance. J Biol Chem 283, 21220-21229.
20.Izadpanah, R., Trygg, C., Patel, B., Kriedt, C., Dufour, J., Gimble, J. M., and Bunnell, B. A. (2006). Biologic properties of mesenchymal stem cells derived from bone marrow and adipose tissue. J Cell Biochem 99, 1285-1297.
21.Jaiswal, N., Haynesworth, S. E., Caplan, A. I., and Bruder, S. P. (1997). Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 64, 295-312.
22.Michalik, L., Desvergne, B., and Wahli, W. (2004). Peroxisome-proliferator-activated receptors and cancers: complex stories. Nat Rev Cancer 4, 61-70.
23.Morrison, R. F., and Farmer, S. R. (1999). Role of PPARgamma in regulating a cascade expression of cyclin-dependent kinase inhibitors, p18(INK4c) and p21(Waf1/Cip1), during adipogenesis. J Biol Chem 274, 17088-17097.
24.Mueller, E., Drori, S., Aiyer, A., Yie, J., Sarraf, P., Chen, H., Hauser, S., Rosen, E. D., Ge, K., Roeder, R. G., and Spiegelman, B. M. (2002). Genetic analysis of adipogenesis through peroxisome proliferator-activated receptor gamma isoforms. J Biol Chem 277, 41925-41930.
25.Muraglia, A., Corsi, A., Riminucci, M., Mastrogiacomo, M., Cancedda, R., Bianco, P., and Quarto, R. (2003). Formation of a chondro-osseous rudiment in micromass cultures of human bone-marrow stromal cells. J Cell Sci 116, 2949-2955.
26.Negishi, Y., Ui, N., Nakajima, M., Kawashima, K., Maruyama, K., Takizawa, T., and Endo, H. (2001). p21Cip-1/SDI-1/WAF-1 gene is involved in chondrogenic differentiation of ATDC5 cells in vitro. J Biol Chem 276, 33249-33256.
27.Nishimura, R., Hata, K., Ikeda, F., Ichida, F., Shimoyama, A., Matsubara, T., Wada, M., Amano, K., and Yoneda, T. (2008). Signal transduction and transcriptional regulation during mesenchymal cell differentiation. J Bone Miner Metab 26, 203-212.
28.Oishi, Y., Manabe, I., Tobe, K., Tsushima, K., Shindo, T., Fujiu, K., Nishimura, G., Maemura, K., Yamauchi, T., Kubota, N., et al. (2005). Kruppel-like transcription factor KLF5 is a key regulator of adipocyte differentiation. Cell Metab 1, 27-39.
29.Ortega, F. J., Vazquez-Martin, A., Moreno-Navarrete, J. M., Bassols, J., Rodriguez-Hermosa, J., Girones, J., Ricart, W., Peral, B., Tinahones, F. J., Fruhbeck, G., et al. (2010). Thyroid hormone responsive Spot 14 increases during differentiation of human adipocytes and its expression is down-regulated in obese subjects. Int J Obes (Lond) 34, 487-499.
30.Owen, H. C., Ahmed, S. F., and Farquharson, C. (2009). Chondrocyte p21(WAF1/CIP1) expression is increased by dexamethasone but does not contribute to dexamethasone-induced growth retardation in vivo. Calcif Tissue Int 85, 326-334.
31.Paul, G., Li, J. Y., and Brundin, P. (2002). Stem cells: hype or hope? Drug Discov Today 7, 295-302.
32.Perez-Castillo, A., Schwartz, H. L., and Oppenheimer, J. H. (1987). Rat hepatic mRNA-S14 and lipogenic enzymes during weaning: role of S14 in lipogenesis. Am J Physiol 253, E536-542.
33.Peroni, D., Scambi, I., Pasini, A., Lisi, V., Bifari, F., Krampera, M., Rigotti, G., Sbarbati, A., and Galie, M. (2008). Stem molecular signature of adipose-derived stromal cells. Exp Cell Res 314, 603-615.
34.Rosen, E. D., and MacDougald, O. A. (2006). Adipocyte differentiation from the inside out. Nat Rev Mol Cell Biol 7, 885-896.
35.Rosen, E. D., Walkey, C. J., Puigserver, P., and Spiegelman, B. M. (2000). Transcriptional regulation of adipogenesis. Genes Dev 14, 1293-1307.
36.Schaffler, A., and Buchler, C. (2007). Concise review: Adipose tissue-derived stromal cells - Basic and clinical implications for novel cell-based therapies. Stem Cells 25, 818-827.
37.Snowden, A. W., Anderson, L. A., Webster, G. A., and Perkins, N. D. (2000). A novel transcriptional repression domain mediates p21(WAF1/CIP1) induction of p300 transactivation. Mol Cell Biol 20, 2676-2686.
38.Soria, G., Speroni, J., Podhajcer, O. L., Prives, C., and Gottifredi, V. (2008). p21 differentially regulates DNA replication and DNA-repair-associated processes after UV irradiation. J Cell Sci 121, 3271-3282.
39.Theodoropoulou, M., Stalla, G. K., and Spengler, D. ZAC1 target genes and pituitary tumorigenesis. Mol Cell Endocrinol 326, 60-65.
40.Tran, T. T., and Kahn, C. R. (2010). Transplantation of adipose tissue and stem cells: role in metabolism and disease. Nat Rev Endocrinol 6, 195-213.
41.Tsuda, T., Markova, D., Wang, H., Evangelisti, L., Pan, T. C., and Chu, M. L. (2004). Zinc finger protein ZAC1 is expressed in chondrogenic sites of the mouse. Dev Dyn 229, 340-348.
42.Valente, T., Junyent, F., and Auladell, C. (2005). ZAC1 is expressed in progenitor/stem cells of the neuroectoderm and mesoderm during embryogenesis: differential phenotype of the ZAC1-expressing cells during development. Dev Dyn 233, 667-679.
43.Van Dyck, F., Declercq, J., Braem, C. V., and Van de Ven, W. J. (2007). PLAG1, the prototype of the PLAG gene family: versatility in tumour development (review). Int J Oncol 30, 765-774.
44.Wu, Z., Xie, Y., Bucher, N. L., and Farmer, S. R. (1995). Conditional ectopic expression of C/EBP beta in NIH-3T3 cells induces PPAR gamma and stimulates adipogenesis. Genes Dev 9, 2350-2363.
45.Zhu, Y., Liu, T., Song, K., Fan, X., Ma, X., and Cui, Z. (2008). Adipose-derived stem cell: a better stem cell than BMSC. Cell Biochem Funct 26, 664-675.
46.Zuk, P. A., Zhu, M., Ashjian, P., De Ugarte, D. A., Huang, J. I., Mizuno, H., Alfonso, Z. C., Fraser, J. K., Benhaim, P., and Hedrick, M. H. (2002). Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 13, 4279-4295.
47.Zuscik, M. J., Hilton, M. J., Zhang, X., Chen, D., and O'Keefe, R. J. (2008). Regulation of chondrogenesis and chondrocyte differentiation by stress. J Clin Invest 118, 429-438.

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top