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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 鄭景暉 | |
dc.contributor.author | Ya-Chin Chang | en |
dc.contributor.author | 張雅芩 | zh_TW |
dc.date.accessioned | 2021-06-15T05:04:13Z | - |
dc.date.available | 2015-09-09 | |
dc.date.copyright | 2010-09-09 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-07-27 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/46336 | - |
dc.description.abstract | 實驗目的:鹼性成纖維細胞生長因子(basic fibroblast growth factor, bFGF)功能包括促進傷口癒合、血管新生、黑色素形成、硬骨及軟骨的生成等…。本篇研究著重於鹼性成纖維細胞生長因子對於人類牙髓細胞在形態表現、細胞增生及生存能力、及細胞移動、細胞分化、細胞間質生成的影響,進而了解bFGF 在牙髓組織修復與重建所扮演的角色。
實驗方法:使用不同濃度的鹼性成纖維細胞生長因子(bFGF)刺激人類牙髓細胞。在不同的時間點下,以光學顯微鏡觀察細胞型態及移動情況。利用MTT 測定牙髓細胞的存活能力;以鹼性磷酸酶染色檢測細胞分化程度、以Sircol Collgen assay 做膠原蛋白定量。另外使用反轉錄鏈聚合反應(RT-PCR)印證相關基因的表現。 實驗結果:鹼性成纖維細胞生長因子(bFGF)會增加細胞數目;人類牙髓細胞在較高濃度的bFGF 刺激下,細胞的存活能力有兩倍至三倍的提升。而與細胞增生相關的基因(Cyclin B1, CDC2, CDC25c)表現會因應bFGF 濃度而增加。bFGF 亦促進牙髓細胞的傷口癒合及細胞移動速度。然而,Rho 和ROCK 基因的表現最高峰會落在bFGF 濃度為10 ng/ml 之上;a-sma 則因應bFGF 濃度而遞減。在細胞分化方面,bFGF 會降低鹼性磷酸酶的染色程度。在細胞間質生成方面,bFGF 對於膠原蛋白的含量沒有顯著影響;而相關基因(TIMP2, MMP2)則因應bFGF 刺激而下降。 結論:人類牙髓細胞受到短期bFGF 刺激後(五天內),細胞增生、移動、傷口癒合能力會因應生長因子而有所增進。然而,bFGF 會影響細胞分化及膠原蛋白的形成。 | zh_TW |
dc.description.abstract | Aim: Basic fibroblast growth factor (bFGF) is multifunctional protein with a wide variety of effects. The functions of bFGF include wound healing, angiogenesis, melanogenesis, development of bone and cartilage, and so on. The purpose of our study is to investigate whether basic fibroblast growth factor influences the morphological changes, cell proliferation and viability, cell differentiation, and extracellular matrix formation of human dental pulp cells in vitro within the period of 5 days.
Materials and Methods: Primary-cultured human dental pulp cells were treated with different concentrations of bFGF (0, 1, 10, 50, 200 ng/ml). Morphology of pulp cells and cell migration were observed under light microscopy (40X). Cell proliferation was evaluated by MTT assay. Cell differentiation was evaluated by alkaline phosphatase (ALP) staining. Changes in mRNA expression of ALKP, Runx2, Cyclin B1, CDC2, CDC25c, p21, Rho, ROCK, a-sma, collagen type I, TIMP2, and MMP2 were determined by reverse-transcriptase polymerase chain reaction (RT-PCR). Collagen content was determined by Sircol Collagen assay. Results: Dental pulp cells are spindle with extended cellular processes with/without bFGF treatment. The numbers of dental pulp cells without any bFGF treatment were much lower than those under the treatment of various concentrations of bFGF. Cell viability was two to three fold increased in higher concentrations of bFGF (50, 200 ng/ml). bFGF increased Cyclin B1, CDC2, and CDC25C mRNA gene expressions dose-dependently. Migrating rates were higher in the groups of higher concentrations of bFGF than those in the groups of lower concentrations. Rho and ROCK gene expressions were at peak at 10 ng/ml of bFGF. Nevertheless, α-sma mRNA gene expression of pulp cells declined in dose-dependent manner. bFGF downregulated ALPactivity of human dental pulp cells but did not affect significantly RUNX2 mRNA expression. bFGF did not affect significantly collagen content . Gene expression of MMP2 and TIMP2 were down-regulated by bFGF treatment. Conclusion: Human dental pulp cells incubated with basic fibroblast growth factor demonstrated proliferative and migratory properties during the early stage of wound repairing (within 5 days). Meanwhile, bFGF had inverse effects on cell differentiation and extracellular matrix formation. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T05:04:13Z (GMT). No. of bitstreams: 1 ntu-99-R96422020-1.pdf: 17572228 bytes, checksum: 9cc934de1804b0b52af7218fff08ef54 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | 中文摘要......................................................................................... i
Abstract ......................................................................................... 2 Chapter I. Literature Review...................................................... 4 1.1.Basic Fibroblast Growth Factor (bFGF, FGF2).................................................. 6 1.1.1. General concept of FGF family.............................................................. 6 1.1.2. Fibroblast Growth Factors Isoforms, Localization and Their Peptide Structure............ 6 1.1.3. The Biological Effects of Basic Fibroblast Growth factor (bFGF) ........ 8 1.1.4. The Role of Fibroblast Growth Factors in tooth development and differentiation ............9 1.1.5. Basic Fibroblast Growth Factors in Dental Pulp Tissue........................11 1.2. The role of Cyclin-Dependent Kinases 1 (CDK1), CDC25C, Cyclin B1, p21 in Cell Proliferation ................ 14 1.2.1. Cell cycle .................................... 14 1.2.2. The Role of Cyclins and Cyclin-Dependent Kinases (CDKs) ............. 16 1.2.3. Inhibitors....................................... 19 1.2.4. CDC25C .................................... 20 1.3. The role of Rho and ROCK in cell migration .................................... 20 1.3.1. Rho GTPases ............................ 20 1.3.2. Rho proteins and cell motility ................ 21 1.4. The role of alpha smooth muscle actin (-sma) in cell contractility ............... 23 Chapter II. The purpose of the study....................................... 25 Chapter III. Materials & Methods ........................ 26 3.1. Materials ..................................... 26 3.2. Culture of human dental pulp cells.................. 26 3.3. Morphology of human dental pulp cells.......................................................... 27 3.4. MTT assay ................................... 28 3.5. Wound healing assay and cell migration assay................................................ 29 3.6. Alkaline phosphatase (ALP) staining ................30 3.7. Quantitative assay of Alkaline phosphatase activity ....................................... 31 3.7.1.Cell lysate collection ................. 31 3.7.2. Alkaline phosphatase activity measurement......................................... 31 3.8. Collagen content assay .............................32 3.9. Reverse Transcription Polymerase Chain Reaction (RT-PCR) ....................... 34 3.9.1. Isolation of total RNA ........................ 35 3.9.2. RNA Quantitation...................... 36 3.9.3. Reverse Transcription (RT) ................ 36 3.9.4. Polymerase Chain Reaction (PCR).................. 37 3.10. Immunofluorescence assay............................... 39 Chapter IV. Results .....................42 4.1. Morphological observation of human dental pulp cells .................................. 42 4.2. Effects of bFGF on cell viability of human dental pulp cells—MTT assay.... 42 4.3. Effects of bFGF on wound healing and cell migration of human dentalpulp cells—wound healing assay and cell migration assay............................................ 43 4.4. Effects of bFGF on alkaline phosphatase (ALP) activity of pulp cells—ALP staining and ALP activity quantitative assay.......................................................... 44 4.5. Effects of bFGF on ALP and Runx2 mRNA expression of pulp cells ............ 45 4.6. Effects of bFGF on collagen formation of dental pulp cells—Sircol assay .... 46 4.7. Effects of bFGF on collagen type I, MMP2 and TIMP2 mRNA expression of pulp cells—RT-PCR ............................................................................................... 46 4.8. Effects of bFGF on Cyclin B1, CDK1 (cdc2), cdc25c, and p21 mRNA expression of pulp cells—RT-PCR......................................................................... 47 4.9. Effects of bFGF on Rho, ROCK, -sma mRNA expression of pulp cells—RT-PCR ....................................................................................................... 48 4.10. Immunofluorescence for mitotic assay.......................................................... 49 Chapter V. Discussion ................................................................ 51 5.1. Effects of bFGF on cell viability and proliferation of human dental pulp cells........................ 51 5.2. Effects of bFGF on cell migration and wound healing of human dental pulp cells........................ 54 5.3. Effect of bFGF on cell differentiation of human dental pulp cells.................. 57 5.4. Effect of bFGF on matrix production of human dental pulp cells................... 60 Chapter VI. Conclusion........................... 62 References ..............................64 | |
dc.language.iso | en | |
dc.title | 鹼性成纖維細胞生長因子對牙髓之增生、移動、分化及細胞間質生成的影響 | zh_TW |
dc.title | Effects of basic fibroblast growth factor on proliferation, migration, differentiation, and matrix production of human dental pulp cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張美姬,林俊彬,陳朝寶 | |
dc.subject.keyword | 牙髓細胞,鹼性成纖維細胞生長因子,細胞增生及移動, | zh_TW |
dc.subject.keyword | dental pulp cells,bFGF,cell proliferation and migration, | en |
dc.relation.page | 108 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-07-28 | |
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 |
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