請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54484
完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 鄭景暉(Jiang-Huei Jeng),張曉華(Hsiao-Hua Chang) | |
dc.contributor.author | Chih-Yu Chen | en |
dc.contributor.author | 陳芝宇 | zh_TW |
dc.date.accessioned | 2021-06-16T02:59:39Z | - |
dc.date.available | 2018-09-24 | |
dc.date.copyright | 2015-09-24 | |
dc.date.issued | 2015 | |
dc.date.submitted | 2015-07-04 | |
dc.identifier.citation | Abe S, Yamaguchi S, Amagasa T (2007). Multilineage cells from apical pulp of human tooth with immature apex. Oral Sci Int 4:45-58.
Arpino V, Brock M, Gill SE (2015). The role of TIMPs in regulation of extracellular matrix proteolysis. Matrix Biol pii: S0945-053X(15)00056-6. Beenken A, Mohammadi M (2009). The FGF family: biology, pathophysiology and therapy. Nat Rev Drug Discov 8:235-53. Review Bikfalvi A, Klein S, Pintucci G, Rifkin DB (1997). Biological Roles of Fibroblast Growth Factor-2. Endocr Rev 18:26-45. Review. Brooks AN, Kilgour E, Smith PD (2012). Molecular pathways: fibroblast growth factor signaling: a new therapeutic opportunity in cancer Clin Cancer Res 18:1855-62. Bruderer M, Richards RG, Alini M, Stoddart MJ (2014). Role and regulation of Runx2 in osteogenesis. Eur Cell Mater 28:269-86. Camilleri S, McDonald F (2006). Runx2 and dental development. Eur J Oral Sci. 114:361-73. Catela C, Bilbao-Cortes D, Slonimsky E, Kratsios P, Rosenthal N, Te Welscher P (2009). Multiple congenital malformations of Wolf-Hirschhorn syndrome are recapitulated in Fgfrl1 null mice. Dis Model Mech 2:283–294. Chambard JC, Lefloch R, Pouysségur J, Lenormand P (2007). ERK implication in cell cycle regulation. Biochim Biophys Acta 1773:1299-310. Chen Q, Li WJ, Wan YY, Yu CD, Li WG (2012). Fibroblast growth factor receptor 4 Gly388Arg polymorphism associated with severity of gallstone disease in a Chinese population. Genet Mol Res 11:548–555. Coutu DL, Galipeau J (2011). Roles of FGF signaling in stem cell self-renewal, senescence and aging. Aging (Albany NY) 3:920-33. Dailey L, Ambrosetti D, Mansukhani A, Basilico C (2005). Mechanisms underlying differential responses to FGF signaling. Cytokine Growth Factor Rev. 16:233-47. Dombrowski C, Helledie T, Ling L, Grünert M, Canning CA, Jones CM, Hui JH, Nurcombe V, van Wijnen AJ, Cool SM (2013). FGFR1 signaling stimulates proliferation of human mesenchymal stem cells by inhibiting the cyclin-dependent kinase inhibitors p21Waf1 and p27Kip1. Stem Cells 31:2724-36. Elfenbein A, Simons M (2013). Syndecan-4 signaling at a glance. J Cell Sci 126 (Pt 17):3799-804. Eswarakumar VP, Lax I, Schlessinger J (2005). Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev 16(2):139-49. Franceschi RT, Xiao G (2003). Regulation of the osteoblast-specific transcription factor, Runx2: responsiveness to multiple signal transduction pathways. J Cell Biochem 88:446-54. Goldberg M, Kulkarni AB, Young M, Boskey A (2011). Dentin: Structure, Composition and Mineralization. Front Biosci (Elite Ed). 3:711-35. Gong SG (2014). Isoforms of receptors of fibroblast growth factors. J Cell Physiol. 229:1887-95. Hakki SS, Hakki EE, Nohutcu RM (2009). Regulation of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases by basic fibroblast growth factor and dexamethasone in periodontal ligament cells. J Periodontal Res 44:794-802. He H, Yu J, Liu Y, Lu S, Liu H, Shi J, Jin Y (2008). Effects of FGF2 and TGFbeta1 on the differentiation of human dental pulp stem cells in vitro. Cell Biol lnt 32:827–34. Huang GT, Sonoyama W, Liu Y, Liu H, Wang S, Shi S (2008). The hidden treasure in apical papilla: the potential role in pulp/dentin regeneration and bioroot engineering. J Endod 34: 645-651. Huang GT, Yamaza T, Shea LD, Djouad F, Kuhn NZ, Tuan RS, Shi S (2010). Stem/progenitor cell-mediated de novo regeneration of dental pulp with newly deposited continuous layer of dentin in an in vivo model. Tissue Eng Part A 16: 605-615. Hughes SE (1997). Differential expression of the fibroblast growth factor receptor (FGFR) multigene family in normal human adult tissues. J Histochem Cytochem 45:1005-19. Ishimatsu H, Kitamura C, Morotomi T, Tabata Y, Nishihara T, Chen KK, Terashita M (2009). Formation of dentinal bridge on surface of regenerated dental pulp in dentin defects by controlled release of fibroblast growth factor-2 from gelatin hydrogels. J Endod 35(6):858-65 Johnson GL, Lapadat R (2002). Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 298:1911–1912. Johnston CL, Cox HC, Gomm JJ, Coombes RC (1995). Fibroblast growth factor receptors (FGFRs) localize in different cellular compartments. J Biol Chem 270:30643-50. Kalinina J, Dutta K, Ilghari D, Beenken A, Goetz R, Eliseenkova AV, Cowburn D, Mohammadi M (2012). The alternatively spliced acid box region plays a key role in FGF receptor autoinhibition. Structure 20:77–88. Kardami E, Detillieux K, Ma X, Jiang Z, Santiago JJ, Jimenez SK, Cattini PA (2007). Fibroblast growth factor-2 and cardioprotection. Heart Fail Rev 12:267-77. Review. Kim HJ, Kim JH, Bae SC, Choi JY, Kim HJ, Ryoo HM (2003). The protein kinase C pathway plays a central role in the fibroblast growth factor-stimulated expression and transactivation activity of Runx2. J Biol Chem 278:319-26. Kim YH, Yoon DS, Kim HO, Lee JW (2012). Characterization of different subpopulations from bone marrow-derived mesenchymal stromal cells by alkaline phosphatase expression. Stem Cells Dev 21:2958-68. Kim YS, Min KS, Jeong DH, Jang JH, Kim HW, Kim EC (2010). Effects of fibroblast growthfactor-2 on the expression and regulation of chemokines in human dental pulp cells. J Endod 36(11):1824-30. Komori T (2011). Signaling networks in RUNX2-dependent bone development. J Cell Biochem. 112:750-5. Kreis NN, Louwen F, Yuan J (2015). Less understood issues: p21Cip1 in mitosis and its therapeutic potential. Oncogene 34:1758-1767. Laestander C, Engström W (2014). Role of fibroblast growth factors in elicitation of cell responses. Cell Prolif 47:3-11. Lambert E, Dassé E, Haye B, Petitfrère E (2004). TIMPs as multifacial proteins. Crit Rev Oncol Hematol 49:187-98. Li B, Qu C, Chen C, Liu Y, Akiyama K, Yang R, Chen F, Zhao Y, Shi S (2012). Basic fibroblast growth factor inhibits osteogenic differentiation of stem cells from human exfoliated deciduous teeth through ERK signaling. Oral Dis 18:285-92. Li B, Qu C, Chen C, Liu Y, Akiyama K, Yang R, Chen F, Zhao Y, Shi S (2012). Basic fibroblast growth factor inhibits osteogenic differentiation of stem cells from human exfoliated deciduous teeth through ERK signaling. Oral Dis 18:285-92. Ma DK, Ponnusamy K, Song MR, Ming GL, Song H (2009). Molecular genetic analysis of FGFR1 signalling reveals distinct roles of MAPK and PLCγ1 activation for self-renewal of adult neural stem cells. Mol Brain 8:16. Madan AK, Kramer B (2005). Immunolocalization of fibroblast growth factor-2 (FGF-2) in the developing root and supporting structures of the murine tooth. J Mol Histol 36:171-8. Makino T, Jinnin M, Muchemwa FC, Fukushima S, Kogushi-Nishi H, Moriya C, Igata T, Fujisawa A, Johno T, Ihn H (2010). Basic fibroblast growth factor stimulates the proliferation of human dermal fibroblasts via the ERK1⁄2 and JNK pathways. Br J Dermatol 162:717-23. Marie PJ (2012). Fibroblast growth factor signaling controlling bone formation: An update. Gene 498:1-4. Marie PJ, Debiais F, Haÿ E (2002). Regulation of human cranial osteoblast phenotype by FGF-2, FGFR-2 and BMP-2 signaling. Histol Histopathol 17:877-85. Martens W, Bronckaers A, Politis C, Jacobs R, Lambrichts I (2013). Dental stem cells and their promising role in neural regeneration: an update. Clin Oral Investig 17:1969-1983. Mignatti P, Morimoto T, Rifkin DB (1992). Basic fibroblast growth factor, a protein devoid of secretory signal sequence, is released by cells via a pathway independent of the endoplasmic reticulum-Golgi complex. J Cell Physiol 151:81-93. Miraoui H, Oudina K, Petite H, Tanimoto Y, Moriyama K, Marie PJ (2009). Fibroblast growth factor receptor 2 promotes osteogenic differentiation in mesenchymal cells via ERK1/2 and protein kinase C signaling. J Biol Chem 284:4897-904. Mohammadi M, Dionne CA, Li W, Li N, Spivak T, Honegger AM, Jaye M, Schlessinger J (1992). Point mutation in FGF receptor eliminates phosphatidylinositol hydrolysis without affecting mitogenesis. Nature 358:681–4. Morito A, Kida Y, Suzuki K, Inoue K, Kuroda N, Gomi K, Arai T, Sato T (2009). Effects of basic fibroblast growth factor on the development of the stem cell properties of human dental pulp cells. Arch Histol Cytol 72(1):51-64. Murakami M, Elfenbein A, Simons M (2008). Non-canonical fibroblast growth factor signaling in angiogenesis. Cardiovasc Res 78:223-31. Naski MC, Wang Q, Xu J, Ornitz DM (1996). Graded activation of fibroblast growth factor receptor 3 by mutations causing achondroplasia and thanatophoric dysplasia. Nat Genet 13:233–237. Nilsson I, Hoffmann I (2000). Cell cycle regulation by the Cdc25 phosphatase family. Prog Cell Cycle Res 4:107-14. Nugent MA, Iozzo RV (2000). Fibroblast growth factor-2. Int J Biochem Cell Biol 32:115-20. Review. Ornitz DM, Itoh N (2015). The Fibroblast Growth Factor signaling pathway. Wiley Interdiscip Rev Dev Biol Mar 13. Ornitz DM, Xu J, Colvin JS, McEwen DG, MacArthur CA, Coulier F, Gao G, Goldfarb M (1996). Receptor specificity of the fibroblast growth factor family. J Biol Chem 271(25):15292-7. Orr-Urtreger A, Bedford MT, Burakova T, Arman E, Zimmer Y, Yayon A, Givol D, Lonai P (1993). Developmental localization of the splicing alternatives of fibroblast growth factor receptor-2 (FGFR2). Dev Biol 158, 475–486. Osathanon T, Nowwarote N, Manokawinchoke J, Pavasant P (2013). bFGF and JAGGED1 regulate alkaline phosphatase expression and mineralization in dental tissue‐derived mesenchymal stem cells. J Cell Biochem 114:2551-61. Palosaari H, Pennington CJ, Larmas M, Edwards DR, Tjäderhane L, Salo T (2003). Expression profile of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs in mature human odontoblasts and pulp tissue Eur J Oral Sci 111:117-27. Park JB (2014). Effects of the combination of fibroblast growth factor-2 and bone morphogenetic protein-2 on the proliferation and differentiation of osteoprecursor cells. Adv Clin Exp Med 23:463-7. Powers CJ, McLeskey SW, Wellstein A (2000). Fibroblast growth factors, their receptors and signaling. Endocr Relat Cancer 7:165-97. Review. Qian J, Jiayuan W, Wenkai J, Peina W, Ansheng Z, Shukai S, Shafei Z, Jun L, Longxing N (2014). Basic fibroblastic growth factor affects the osteogenic differentiation of dental pulp stem cells in a treatment-dependent manner. Int Endod J doi: 10.1111/iej.12368. Qiu W, Leibowitz B, Zhang L, Yu J (2010). Growth factors protect intestinal stem cells from radiation induced apoptosis by suppressing PUMA through the PI3K/AKT/ p53 axis. Oncogene 29:1622-32. Ramasamy R, Tong CK, Yip WK, Vellasamy S, Tan BC, Seow HF (2012). Basic fibroblast growth factor modulates cell cycle of human umbilical cord-derivedmesenchymal stem cells. Cell Prolif 45:132-9. Ruparel NB, de Almeida JF, Henry MA, Diogenes A (2013). Characterization of a stem cell of apical papilla cell line: effect of passage on cellular phenotype. J Endod 39: 357-363. Russo LG, Maharajan P, Maharajan V (1998). Basic fibroblast growth factor (FGF-2) in mouse tooth morphogenesis. Growth Factors 15(2):125-33. Rutland P, Pulleyn LJ, Reardon W, Baraitser M, Hayward R, Jones B, Malcolm S, Winter RM, Oldridge M, Slaney SF (1995). Identical mutations in the FGFR2 gene cause both Pfeiffer and Crouzon syndrome phenotypes. Nat Genet 9:173–176. Saito A, Higuchi I, Nakagawa M, Saito M, Uchida Y, InoseM, Kasai T, Niiyama T, Fukunaga H, Arimura K (2000). An overexpression of fibroblast growth factor (FGF) and FGF receptor 4 in a severe clinical phenotype of facioscapulohumeral muscular dystrophy. Muscle Nerve 23:490–497. Shimabukuro Y, Ueda M, Ozasa M, Anzai J, Takedachi M, Yanagita M, Ito M, Hashikawa T, Yamada S, Murakami S (2009). Fibroblast growth factor–2 regulates the cell function of human dental pulp cells. J Endod 35:1529-35. Silverio-Ruiz KG, Martinez AE, Garlet GP, Barbosa CF, Silva JS, Cicarelli RM, Valentini SR, Abi-Rached RS, Junior CR (2007). Opposite effects of bFGF and TGF-β on collagen metabolism by human periodontal ligament fibroblasts. Cytokine 39:130-7. Sonoyama W, Liu Y, Fang D, Yamaza T, Seo BM, Zhang C, Liu H, Gronthos S, Wang CY, Wang S, Shi S (2008). Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: a pilot study. J Endod 34: 166-171. Sonoyama W, Liu Y, Yamaza T, Tuan RS, Wang S, Shi S, Huang GT (2006). Mesenchymal stem cell-mediated functional tooth regeneration in swine. PLoS One.1: e79. S?rensen V, Nilsen T, Wiedłocha A (2006). Functional diversity of FGF-2 isoforms by intracellular sorting. Bioessays 28:504-14. Review. Stein GS, Lian JB, Owen TA (1990). Relationship of cell growth to the regulation of tissue-specific gene expression during osteoblast differentiation. FASEB J. 4:3111-23. Suzuki A, Guicheux J, Palmer G, Miura Y, Oiso Y, Bonjour JP, Caverzasio J (2002). Evidence for a role of p38 MAP kinase in expression of alkaline phosphatase during osteoblastic cell differentiation. Bone 30:91-8. Tobias Duarte PC, Gomes-Filho JE, Ervolino E, Marçal Mazza Sundefeld ML, Tadahirowayama M, Lodi CS, Dezan-Júnior E, Angelo Cintra LT (2014). Histopathological condition of the remaining tissues after endodontic infection of rat immature teeth. J Endod 40:538-42. Touriol C, Bornes S, Bonnal S, Audigier S, Prats H, Prats AC, Vagner S (2003). Generation of protein isoform diversity by alternative initiation of translation at non-AUG codons. Biol Cell 95:169–178. Tsuboi T, Mizutani S, Nakano M, Hirukawa K, Togari A (2003). FGF-2 regulates enamel and dentine formation in mouse tooth germ. Calcif Tissue Int 73(5):496-501. Vermeulen K, Van Bockstaele DR, Berneman ZN (2003). The cell cycle: a review of regulation, deregulation and therapeutic targets incancer. Cell Prolif 36:131-49. Viale-Bouroncle S, Gosau M, Morsczeck C (2014). Collagen I induces the expression of alkaline phosphatase and osteopontin via independent activations of FAK and ERK signalling pathways. Arch Oral Biol 59:1249-55. Vimalraj S, Arumugam B, Miranda PJ, Selvamurugan N (2015). Runx2: Structure, function, and phosphorylation in osteoblast differentiation. Int J Biol Macromol 78:202-208. Walsh S, Margolis SS, Kornbluth S (2003). Phosphorylation of the cyclin B1 cytoplasmic retention sequence by mitogen-activated protein kinase and Plx. Mol Cancer Res 1:280-9. Wright JH, Munar E, Jameson DR, Andreassen PR, Margolis RL, Seger R, Krebs EG (1999). Mitogen-activated protein kinase kinase activity is required for the G(2)/M transition of the cell cycle in mammalian fibroblasts. Proc. Natl. Acad. Sci. U. S. A. 96:11335–11340. Wu IH (2011). Effect of TGF-β1 on the growth and differentiation of human apical papilla cells. Wu J, Huang GT, He W, Wang P, Tong Z, Jia Q, Dong L, Niu Z, Ni L (2012). Basic fibroblast growth factor enhances stemness of human stem cells from the apical papilla. J Endod 38:614-622. Wu Y, Yang Y, Yang P, Gu Y, Zhao Z, Tan L, Zhao L, Tang T, Li Y (2013). The osteogenic differentiation of PDLSCs is mediated through MEK/ERK and p38 MAPK signalling under hypoxia. Arch Oral Biol 58:1357-68. Xiao G, Jiang D, Gopalakrishnan R, Franceschi RT (2002). Fibroblast growth factor 2 induction of the osteocalcin gene requires MAPK activity and phosphorylation of the osteoblast transcription factor, Cbfa1/Runx2. J Biol Chem 277:36181-7. Yang H, Xia Y, Lu SQ, Soong TW, Feng ZW (2008). Basic fibroblast growth factor-induced neuronal differentiation of mouse bone marrow stromal cells requires FGFR-1, MAPK/ERK, and transcription factor AP-1. J Biol Chem 283:5287-95. Yue B (2014). Biology of the extracellular matrix: an overview. J Glaucoma 23:S20-3. Zhang W, Liu HT (2002). MAPK signal pathways in the regulation of cell proliferation in mammalian cells. Cell Res 12:9-18. Zhang X, Ibrahimi OA, Olsen SK, Umemori H, Mohammadi M, Ornitz DM (2006). Receptor Specificity of the Fibroblast Growth Factor Family. The complete mammalian FGF family. J Biol Chem 281:15694-700. Zhao WQ, Li H, Yamashita K, Guo XK, Hoshino T, Yoshida S, Shinya T, Hayakawa T (1998). Cell cycle-associated accumulation of tissue inhibitor of metalloproteinases-1 (TIMP-1) in the nuclei of human gingival fibroblasts. J Cell Sci 111 ( Pt 9):1147-53. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54484 | - |
dc.description.abstract | 實驗目的:鹼性纖維母細胞生長因子(Basic fibroblast growth factor, bFGF)在生物體內具有多種功能,並且在細胞生長與分化上扮演重要角色,但其對於牙根尖細胞之影響及訊息傳導路徑還未被完全了解。本實驗之目的在探討bFGF對牙根尖細胞生長與分化的影響,以及其中MEK/ERK訊息傳導路徑的角色。
實驗方法:加入不同濃度bFGF於人類牙根尖細胞做刺激與培養,部分組別加入U0126 (MEK/ERK抑制劑)。透過細胞存活率分析 (MTT assay)、反轉錄聚合酶連鎖反應 (RT-PCR)、西方墨點法 (western blot)、鹼性磷酸酶染色(ALP stain)及免疫螢光染色(immunofluorescent)來檢測bFGF對牙根尖細胞在細胞生長、分化及相關之基因與蛋白質、纖維母細胞生長因子接受器(FGFR)的影響。 實驗結果:bFGF促進牙根尖細胞生長與相關基因及蛋白之表現,包括cyclin B1、cdc2及cdc25c。在成骨/成牙本質分化方面,經過24小時bFGF刺激後,Runx2和osteocalcin表現會增加,但5天之bFGF刺激則會抑制ALP活性。FGFR1、2、3及4均會表現於牙根尖細胞。單獨加入U0126會抑制細胞生長,而合併bFGF使用會降低原本bFGF促進生長之能力。此外,U0126可以抑制因bFGF提升之osteocalcin與TIMP1基因及蛋白表現,而對Runx2與ALP則無影響。U0126亦無法逆轉因bFGF下降之ALP活性。 結論:bFGF對人類牙根尖細胞的影響是很多樣性的,會因藥物刺激的時間而不同,而MEK/ERK在其中亦扮演很重要的角色。本實驗的結果提供我們對bFGF與牙根尖細胞的作用及訊息傳導路徑更進一步的了解,對於未來應用在牙髓壞死之牙齒的根尖生成術與牙髓、牙本質再生也有幫助。 | zh_TW |
dc.description.abstract | Aim : Basic fibroblast growth factor (bFGF) owns multiple biological functions in various tissues, and plays important roles in cell proliferation and differentiation. Human apical papilla cells have been reported to show characteristics of stem cells and are known as stem cells from apical papilla (SCAP). The purpose of this study is to investigate the effects of bFGF on apical papilla cells and the roles of MEK/ERK signaling. We hypothesize that bFGF regulates cell proliferation and differentiation through MEK/ERK.
Materials and Methods : Primary-cultured human apical papilla cells were treated under different concentration with or without U0126 (an inhibitor of MEK/ERK). Cell proliferation was measured by MTT assay. The expressions of cell cycle-related and differentiation-related genes and proteins were examined by reverse transcription polymerase chain reaction (RT-PCR) and western blot, respectively. Phosphorylation of signaling molecules was examined by western blot. ALP activity was determined by ALP staining. FGF receptors (FGFRs) were detected by immunofluorescent (IF). Results : In human apical papilla cells, treatment of bFGF (10~500 ng/ml) enhanced the proliferation and the expression of cell cycle-related genes and proteins including cyclin B1, cdc2, and cdc25c. In osteogenic/ dentinogenic differentiation, bFGF promoted the expression of Runx2 and osteocalcin, but ALP activity was suppressed by treatment of bFGF for 5 days. FGFR1, 2, 3 and 4 were expressed abundantly in apical papilla cells. Using U0126 solely decreased the inherent proliferative ability in apical papilla cells, and combined with bFGF inhibited the bFGF-induced enhancement of proliferation and expression of cell cycle-related genes and proteins. The increase of osteocalcin and TIMP1 induced by bFGF was repressed by U0126, while Runx2 and ALP were not changed. Besides, ALP activity attenuated by bFGF could not be reversed by U0126. Conclusion : The effect of bFGF on apical papilla cells is complicated and might be divergent depending on the duration of treatment. MEK/ERK pathway plays important roles in miscellaneous cell functions. These results would be useful for clinical therapies in the future, including apexogenesis and pulp-dentin complex regeneration. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T02:59:39Z (GMT). No. of bitstreams: 1 ntu-104-R01422026-1.pdf: 2902662 bytes, checksum: 643325e5a1b93c45e9405018e38a90b7 (MD5) Previous issue date: 2015 | en |
dc.description.tableofcontents | 口試委員會審定書 i
中文摘要 ii Abstract iv Chapter I Literature Review 1 1.1 Stem cells from apical papilla 1 1.2 Basic fibroblast growth factor 3 1.2.1 The structure of bFGF and isoforms 4 1.2.2 Receptors of bFGF 5 1.2.3 Canonical signal transduction pathways of bFGF 6 1.2.4 Non-canonical signal transduction pathways of bFGF 7 1.2.5 The effect of bFGF on tooth development and dental cells 8 1.3 Cell cycle 9 1.3.1 Cyclin-dependent kinase (CDK) regulation 9 1.3.2 Cyclin-dependent kinase (CDK) inhibition 10 1.4 Extracellular matrix (ECM) 10 1.5 Alkaline phosphatase (ALP) 11 1.6 Runt-related transcription factor 2 (Runx2) 12 Chapter II The Purposes and Hypothesis of this Study 13 Chapter III Materials and Methods 14 3.1 Culture of human apical papilla cells 14 3.2 Surface marker of apical papilla cells 14 3.3 MTT assay 15 3.4 Immunofluorescent (IF) microscope observation 15 3.5 Reverse transcription polymerase chain reaction (RT-PCR) 16 3.5.1 Isolation of total RNA 17 3.5.2 RNA Quantitation 18 3.5.3 Reverse Transcription (RT) 18 3.5.4 Polymerase chain reaction (PCR) 19 3.6 Western blot 19 3.6.1 Protein extraction 20 3.6.2 Protein quantification 20 3.6.3 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis 21 3.6.4 Transfer Gel 21 3.6.5 Blocking and Antibody Hybridization 22 3.6.6 Chemiluminescence photography 22 3.7 Alkaline phosphatase stain (ALP stain) 23 3.8 Statistical analysis 23 Chapter IV Results 25 4.1 Morphological observation on human apical papilla cells 25 4.2 Surface marker of apical papilla cells 25 4.3 Effects of bFGF on cell proliferation of apical papilla cells - MTT assay 25 4.4 Effect of bFGF on cell cycle-related genes: focus on G2/M phase 25 4.5 Effect of bFGF on cell cycle-related proteins: focus on G2/M phase 26 4.6 Effect of bFGF on osteogenic/dentinogenic differentiation-related genes and proteins of apical papilla cells – RTPCR and western blot 26 4.7 Effect of bFGF on ALP activity of apical papilla cells – ALP stain 26 4.8 Effect of bFGF on ECM-related genes and proteins in apical papilla cells 26 4.9 Effect of bFGF on FGF receptors (FGFR) expression in apical papilla cells - ImmunoFluorescence (IF) 27 4.10 The signaling pathways induced by bFGF in apical papilla cells – western blot……………………………………………………………………………...27 4.11 Effects of U0126 on cell proliferation induced by bFGF of apical papilla cells - MTT assay 27 4.12 Effect of U0126 on cell cycle-related genes induced by bFGF: focus on G2/M phase 28 4.13 Effect of U0126 on cell cycle-related proteins induced by bFGF: focus on G2/M phase 28 4.14 Effect of U0126 on osteogenic/dentinogenic differentiation-related genes and proteins induced by bFGF – RTPCR and western blot 28 4.15 Effect of U0126, LY294002, and H7 on ALP activity induced by bFGF– ALP stain 29 4.16 Effect of U0126 on ECM-related genes and proteins induced by bFGF – RTPCR and western blot 29 Chapter V Discussion 30 5.1 Effect of bFGF on cell proliferation and cell cycle-related genes and proteins of human apical papilla cells 30 5.2 Effect of bFGF on cell differentiation of human apical papilla cells 31 5.3 Effect of bFGF on expression of FGFRs of human apical papilla cells 33 5.4 Effect of U0126 on cell proliferation and cell cycle-related genes and proteins of human apical papilla cells 34 5.5 Effect of U0126 on cell differentiation and related genes and proteins of human apical papilla cells 35 Chapter VI Conclusion 38 References 39 Figures Figure 1: Apical papilla (I-Hua Wu, 2011) 49 Figure 2: Apical papilla cells 49 Figure 3: Cell morphology of apical papilla cells (100X) 50 Figure 4: Cell surface markers (I-Hua Wu, 2011) 51 Figure 5: MTT assay: bFGF 0~ 500 ng/ml in serum-free medium 52 Figure 6: MTT assay: bFGF 0~ 500 ng/ml in medium containing serum 53 Figure 7: Effect of bFGF on mRNA expression of cell cycle-related genes 54 Figure 8: Effect of bFGF on expression of cell cycle-related proteins 55 Figure 9: Effect of bFGF on expression of differentiation-related genes 56 Figure 10: Effect of bFGF on expression of differentiation-related proteins 57 Figure 11: ALP stain 58 Figure 12: Effect of bFGF on mRNA expression of ECM-related genes 59 Figure 13: Effect of bFGF on expression of ECM-related proteins 60 Figure 14: FGFR1 expression in apical papilla cells 61 Figure 15: FGFR2 expression in apical papilla cells 62 Figure 16: FGFR3 expression in apical papilla cells 63 Figure 17: FGFR4 expression in apical papilla cells 64 Figure 18: Location of FGFRs in the apical papilla cells (400X) 65 Figure 19: Signaling pathways activated by bFGF (250 ng/ml) 66 Figure 20: MTT assay: bFGF 250 ng/ml with U0126 (10, 20 μM) in serum-free medium 67 Figure 21: MTT assay: bFGF 250 ng/ml with U0126 (10, 20 μM) in medium with serum. 68 Figure 22: Effect of bFGF (250 ng/ml) with/without U0126 (10, 20 μM) on mRNA expression of cell cycle-related genes 69 Figure 23: Effect of bFGF (250 ng/ml) with/without U0126 (10, 20 μM) on expression of cell cycle-related proteins 70 Figure 24: Effect of bFGF (250 ng/ml) with/without U0126 (10, 20 μM) on expression of osteogenic/odontogenic differentiation-related genes 71 Figure 25: Effect of bFGF (250 ng/ml) with/without U0126 (10, 20 μM) on expression of osteogenic/odontogenic differentiation-related proteins 72 Figure 26: ALP stain with bFGF and U0126 73 Figure 27: ALP stain with bFGF and LY294002 74 Figure 28: ALP stain with bFGF and H7 75 Figure 29: Effect of bFGF (250 ng/ml) with/without U0126 (10, 20 μM) on expression of ECM-related genes 76 Figure 30: Effect of bFGF (250 ng/ml) with/without U0126 (10, 20 μM) on expression of ECM-related proteins 77 Tables Table 1. Sequences and base pairs of PCR primers 78 Table 2. Protocols of Western Blot protein extraction buffer 79 Table 3a. Protocols of resolving gel for SDS-PAGE 79 Table 3b. Protocols of stacking gel for SDS-PAGE 79 Table 4. Protocol for Western Blot SDS-PAGE running buffer 80 Table 5. Protocol for Western Blot SDS-PAGE trasnfer buffer 80 Table 6. Molecular weight, host and concentration of western blot primary antibodies 81 Table 7. Protocol for Western Blot Tween TBS 83 Table 8. MTT assay: bFGF 0~500 ng/ml in serum-free medium 84 Table 9. MTT assay: bFGF 0~500 ng/ml in medium with serum 85 Table 10. MTT assay: bFGF 250 ng/ml with U0126 (10, 20 μM) in serum-free medium 86 Table 11. MTT assay: bFGF 250 ng/ml with U0126 (10, 20 μM) in medium with serum…….……………………………………………………………………..87 | |
dc.language.iso | zh-TW | |
dc.title | bFGF對於牙根尖細胞生長及分化之影響:MEK/ERK訊息傳導路徑的角色 | zh_TW |
dc.title | Effect of bFGF on the growth and differentiation of human apical papilla cells: Role of MEK/ERK signaling pathway | en |
dc.type | Thesis | |
dc.date.schoolyear | 103-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張美姬(Mei-Chi Chang),李勝揚(Sheng-Yang Lee),楊淑芬(Shue-Fen Yang) | |
dc.subject.keyword | 牙根尖細胞,鹼性纖維母細胞生長因子,纖維母細胞生長因子接受器,MEK/ERK,鹼性磷酸?, | zh_TW |
dc.subject.keyword | apical papilla cells,bFGF,FGFR,MEK/ERK,alkaline phosphatase, | en |
dc.relation.page | 87 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2015-07-06 | |
dc.contributor.author-college | 牙醫專業學院 | zh_TW |
dc.contributor.author-dept | 臨床牙醫學研究所 | zh_TW |
顯示於系所單位: | 臨床牙醫學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-104-1.pdf 目前未授權公開取用 | 2.83 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。