進階搜尋


   電子論文尚未授權公開,紙本請查館藏目錄
(※如查詢不到或館藏狀況顯示「閉架不公開」,表示該本論文不在書庫,無法取用。)
系統識別號 U0026-1907201609192900
論文名稱(中文) 從牙齦纖維母細胞探討Nifedipine造成牙齦增生的分子機制
論文名稱(英文) The molecular mechanisms of nifedipine-associated gingival overgrowth in gingival fibroblasts
校院名稱 成功大學
系所名稱(中) 口腔醫學研究所
系所名稱(英) Institute of Oral Medicine
學年度 104
學期 2
出版年 105
研究生(中文) 余金璇
研究生(英文) Jin-Hsuan Yu
學號 T46031020
學位類別 碩士
語文別 中文
論文頁數 66頁
口試委員 指導教授-袁國
口試委員-林英助
口試委員-劉景勳
中文關鍵字 nifedipine  牙齦增生  幹細胞基因 
英文關鍵字 nifedipine  gingival overgrowth  stem cell genes 
學科別分類
中文摘要 Nifedipine為一鈣離子阻斷劑,常廣泛用於治療高血壓及心血管疾病。然而,nifedipine有造成藥物性牙齦增生的副作用,容易去影響病人的口腔衛生、外觀等等。由於目前尚未找到能夠解決這個副作用的辦法,因此去了解nifedipine造成牙齦增生的分子機制就會顯得重要。目前大部分的研究都是著重在nifedipine作用在牙齦纖維母細胞時,其細胞因子或生長因子相關的表現,而較少研究透過幹細胞相關的基因來了解nifedipine造成牙齦增生上的分子機制。因此,我們利用human stem cell PCR array去預測標的基因,透過分子機制層面來了解藥物對牙齦纖維母細胞影響情形。我們首先用含有不同藥物濃度的培養基去培養牙齦纖維母細胞,分別培養一天、三天、五天、七天共四個時間點,從細胞存活率分析以及即時偵測細胞分析可知,在200 ng/ml的藥物濃度下,對細胞沒有毒殺性的影響,並在第七天細胞有增加的趨勢,利用CD44的mRNA表現量比較下,雖然與控制組比較沒有顯著的差異,但是在第七天相對其他天數有較高的表現,因此,我們最終選擇第七天作為時間點來做後續實驗。透過human stem cell PCR array結果,我們選出有相對明顯差異的基因作探討,包括ALDH2、COL2A1、FOXA2、WNT1,收集更多不同來源的細胞,透過即時定量聚合酶連鎖反應再次驗證mRNA表現,利用西方墨點法及免疫組織化學染色去觀察蛋白質分布,最後我們發現COL2A1在上述實驗中有相對明顯的表現,因此推論nifedipine造成牙齦增生的情形,COL2A1可能參與其中。
英文摘要 To solve the side-effect of nifedipine, it is important to realize the molecular mechanisms of nifedipine-induced gingival overgrowth. Mostly previous studies focus the cytokines or growth factors expressions on nifedipine treatment in fibroblasts, but there are few studies about stem cell markers in the mechanism of nifedipine-associated gingival overgrowth in gingival fibroblasts. Therefore, we cultured healthy gingival fibroblasts with different concentration of nifedipine medium and used human stem cell PCR array to predict target genes, western blots and immunohistochemistry to confirm the proteins expression. We found that 200 ng/mL concentration of DMSO and nifedipine did not influence on gingival fibroblasts proliferation. We also found that CD44 expressions of both groups were upregulated gradually as the time went. Therefore, we cultured the cells for 7 days to run human stem cell PCR array. PCR array results showed that most of genes were upregulated in gingival fibroblasts cultured in nifedipine-treated medium in heat map, but most gene changes were limited to the threshold of 2 folds. We collected more sources of gingival fibroblasts and chose the top of four genes to confirm the mRNA expressions and protein levels, including ALDH2、COL2A1、FOXA2 and WNT1. We found that COL2A1 had significant difference compared to control. We concluded that COL2A1 may participate in nifedipine-induced gingival overgrowth.
論文目次 中文摘要 I
Abstract II
致謝 V
目錄 VI
圖目錄 VIII
英文縮寫檢索表 IX
第一章 緒論 1
一、牙齦 (Gingiva) 1
二、牙齦纖維母細胞(Gingival fibroblasts) 2
三、牙齦增生 (Gingival overgrowth;Gingival enlargement) 2
四、幹細胞標誌 (Stem cell marker) 4
五、研究動機 7
第二章 實驗材料與方法 8
一、免疫化學染色 (Immunohistochemistry) 8
二、SDS-PAGE 蛋白質電泳 ( SDS-PAGE protein electrophoresis ) 12
三、西方墨點法 ( Western blot ) 14
四、細胞培養 ( Cell culture ) 17
五、細胞蛋白質萃取 ( Protein extraction ) 21
六、蛋白質濃度測定 ( Protein assay ) 22
七、萃取RNA ( RNA extraction ) 23
八、反轉錄PCR ( RT-PCR ) 25
九、即時定量PCR ( Real-time PCR ) 26
十、人類幹細胞PCR array ( human stem cell PCR array ) 28
十一、細胞存活率分析 (XTT assay) 29
十二、即時電子式細胞偵測技術 (Real-Time Cell Analyzer Technology, RTCA Technology) 30
十三、統計分析 ( Statistics methods ) 32
十四、儀器設備、試劑藥品、套裝試驗組、抗體、耗材 32
第三章 實驗結果 40
一、利用XTT assay檢測不同藥物濃度對牙齦纖維母細胞生長影響 40
二、利用即時偵測細胞分析儀(RTCA)檢測不同藥物濃度對細胞生長的影響 40
三、利用CD44檢測牙齦纖維母細胞於不同時間點的mRNA表現量 41
四、Human stem cell PCR array分析後有表現差異的基因 41
五、利用Real-time PCR去確認從Human stem cell PCR array中找出有差異性的幹細胞標誌 42
六、比較ALDH2、COL2A1、FOXA2、WNT1在健康牙齦及增生的牙齦兩者蛋白表現量 42
七、比較ALDH2、COL2A1、FOXA2、WNT1在健康牙齦及增生的牙齦組織表現 43
第四章 討論 44
一、Nifedipine造成牙齦增生的機制與幹細胞基因的表現關係 44
二、Nifedipine對牙齦纖維母細胞生長影響 46
三、細胞的選擇 47
四、COL2A1與牙齦增生的關係 47
五、臨床檢體的鑑定蛋白表現 48
第五章 結論 49
參考文獻 50
附圖 54

參考文獻 Bafico, A., Liu, G., Goldin, L., Harris, V., and Aaronson, S.A. (2004). An autocrine mechanism for constitutive Wnt pathway activation in human cancer cells. Cancer Cell 6, 497-506.
Bartold, P.M., Walsh, L.J., and Narayanan, A.S. (2000). Molecular and cell biology of the gingiva. Periodontology 2000 24, 28–55.
Brown, R.S., and Arany, P.R. (2015). Mechanism of drug-induced gingival overgrowth revisited: a unifying hypothesis. Oral Dis 21, e51-e61.
Cai, J., Chen, J., Liu, Y., Miura, T., Luo, Y., Loring, J.F., Freed, W.J., Rao, M.S., and Zeng, X. (2006). Assessing self-renewal and differentiation in human embryonic stem cell lines. Stem Cells 24, 516-530.
Chen, J., Yang, F., Yu, X., Yu, Y., and Gong, Y. (2016). Cyclosporine A promotes cell proliferation, collagen and alpha-smooth muscle actin expressions in rat gingival fibroblasts by Smad3 activation and miR-29b suppression. J Periodontal Res.
Cho, M.-I., and Garant, P.R. (2000). Development and general structure of the periodontium. Periodontology 2000 24, 9–27.
Di, C.P., Sun, Y., Zhao, L., Li, L., Ding, C., Xu, Y., and Fan, Y. (2013). Effect of nifedipine on the expression of keratinocyte growth factor and its receptor in cocultured/monocultured fibroblasts and keratinocytes. J Periodontal Res 48, 740-747.
Ebert, A.D., Kodo, K., Liang, P., Wu, H., Huber, B.C., Riegler, J., Churko, J., Lee, J., de Almeida, P., Lan, F., et al. (2014). Characterization of the molecular mechanisms underlying increased ischemic damage in the aldehyde dehydrogenase 2 genetic polymorphism using a human induced pluripotent stem cell model system. Sci Transl Med 6, 255ra130.
Fournier, B.P., Ferre, F.C., Couty, L., Lataillade, J.J., Gourven, M., Naveau, A., Coulomb, B., Lafont, A., and Gogly, B. (2010). Multipotent progenitor cells in gingival connective tissue. Tissue Eng Part A 16, 2891-2899.
Fujimori, Y., Maeda, S., Saeki, M., Morisaki, I., and Kamisaki, Y. (2001). Inhibition by nifedipine of adherence- and activated macrophage-induced death of human gingival fibroblasts. Eur J Pharmacol 415, 95-103.
Garcı́a-Castro, M.n.I., Marcelle, C., and Bronner-Fraser, M. (2002). Ectodermal Wnt Function as a Neural Crest Inducer. Science 297, 848-851.
Guncu, G.N., Caglayan, F., Dincel, A., Bozkurt, A., Ozmen, S., and Karabulut, E. (2007). Clinical and pharmacological variables as a risk factor for nifedipine-induced gingival overgrowth. Aust Dent J 52, 295-299.
Hunt, J., Brooks, R., Jennions, M.D., Smith, M.J., Bentsen, C.L., and Bussiere, L.F. (2004). High-quality male field crickets invest heavily in sexual display but die young. Nature 432, 1024-1027.
Hyun, J.S., Tran, M.C., Wong, V.W., Chung, M.T., Lo, D.D., Montoro, D.T., Wan, D.C., and Longaker, M.T. (2013). Enhancing stem cell survival in vivo for tissue repair. Biotechnology Advances 31, 736-743.
Ivanova, N., Dobrin, R., Lu, R., Kotenko, I., Levorse, J., DeCoste, C., Schafer, X., Lun, Y., and Lemischka, I.R. (2006). Dissecting self-renewal in stem cells with RNA interference. Nature 442, 533-538.
Jung, J.Y., Jeong, Y.J., Jeong, T.S., Chung, H.J., and Kim, W.J. (2008). Inhibition of apoptotic signals in overgrowth of human gingival fibroblasts by cyclosporin A treatment. Arch Oral Biol 53, 1042-1049.
Kalluri, R., and Zeisberg, M. (2006). Fibroblasts in cancer. Nat Rev Cancer 6, 392-401.
Kantarci, A., Augustin, P., Firatli, E., Sheff, M.C., Hasturk, H., Graves, D.T., and Trackman, P.C. (2007). Apoptosis in gingival overgrowth tissues. J Dent Res 86, 888-892.
Kataoka, M., Kido, J., Shinohara, Y., and Nagata, T. (2005). Drug-induced gingival overgrowth--a review. Biol Pharm Bull 28, 1817-1821.
Kim, S.S., Jackson-Boeters, L., Darling, M.R., Rieder, M.J., and Hamilton, D.W. (2013). Nifedipine induces periostin expression in gingival fibroblasts through TGF-beta. J Dent Res 92, 1022-1028.
Li, Y., Zhang, D., Jin, W., Shao, C., Yan, P., Xu, C., Sheng, H., Liu, Y., Yu, J., Xie, Y., et al. (2006). Mitochondrial aldehyde dehydrogenase-2 (ALDH2) Glu504Lys polymorphism contributes to the variation in efficacy of sublingual nitroglycerin. J Clin Invest 116, 506-511.
Livada, R., and Shiloah, J. (2014). Calcium channel blocker-induced gingival enlargement. J Hum Hypertens 28, 10-14.
Locke, M., Feisst, V., and Dunbar, P.R. (2011). Concise review: human adipose-derived stem cells: separating promise from clinical need. Stem Cells 29, 404-411.
Marcato, P., Dean, C.A., Giacomantonio, C.A., and Lee, P.W. (2011). Aldehyde dehydrogenase: its role as a cancer stem cell marker comes down to the specific isoform. Cell Cycle 10, 1378-1384.
Nakib, N., and Ashrafi, S.S. (2011). Drug-induced gingival overgrowth. Dis Mon 57, 225-230.
Nanci, A., and Bosshardt, D.D. (2006). Structure of periodontal tissues in health and disease. Periodontol 2000 40, 11-28.
Nurmenniemi, P.K., Pernu, H.E., and Knuuttila, M.L. (2001). Mitotic activity of keratinocytes in nifedipine- and immunosuppressive medication-induced gingival overgrowth. J Periodontol 72, 167-173.
Nusse, R., van Ooyen, A., Cox, D., Fung, Y.K.T., and Varmus, H. (1984). Mode of proviral activation of a putative mammary oncogene (int-1) on mouse chromosome 15. Nature 307, 131-136.
Ramon, Y., Behar, S., Kishon, Y., and Engelberg, I.S. (1984). Gingival hyperplasia caused by nifedipine — a preliminary report. International Journal of Cardiology 5, 195-204.
Seymour., R.A., Ellis., J.S., and Thomason., J.M. (1999). Risk factors for drug‐induced gingival overgrowth. J Clin Periodontol 27, 217–223.
Shimizu, Y., Kataoka, M., Seto, H., Kido, J., and Nagata, T. (2002). Nifedipine induces gingival epithelial hyperplasia in rats through inhibition of apoptosis. J Periodontol 73, 861-867.
Sukkar, T.Z., Thomason, J.M., Cawston, T.E., Lakey, R., Jones, D., Catterall, J., and Seymour, R.A. (2007). Gingival fibroblasts grown from cyclosporin-treated patients show a reduced production of matrix metalloproteinase-1 (MMP-1) compared with normal gingival fibroblasts, and cyclosporin down-regulates the production of MMP-1 stimulated by pro-inflammatory cytokines. J Periodontal Res 42, 580-588.
Trackman, P.C., and Kantarci, A. (2004). Connective tissue metabolism and gingival overgrowth. Crit Rev Oral Biol Med 15, 165-175.
Trackman, P.C., and Kantarci, A. (2015). Molecular and clinical aspects of drug-induced gingival overgrowth. J Dent Res 94, 540-546.
Uzel, M.I., Kantarci, A., Hong, H.-H., Uygur, C., Sheff, M.C., Firatli, E., and Trackman, P.C. (2001). Connective tissue growth factor in drug-induced gingival overgrowth. J Periodontol 72, 921-931.
Wang, H.S., Hung, S.C., Peng, S.T., Huang, C.C., Wei, H.M., Guo, Y.J., Fu, Y.S., Lai, M.C., and Chen, C.C. (2004). Mesenchymal stem cells in the Wharton's jelly of the human umbilical cord. Stem Cells 22, 1330-1337.

論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2021-07-20起公開。
  • 同意授權校外瀏覽/列印電子全文服務,於2021-07-20起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw