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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/81211完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭景暉(Jiiang-Huei Jeng),張曉華(Hsiao-Hua Chang) | |
| dc.contributor.author | Tzu-Jung Chen | en |
| dc.contributor.author | 陳姿蓉 | zh_TW |
| dc.date.accessioned | 2022-11-24T03:36:27Z | - |
| dc.date.available | 2021-08-18 | |
| dc.date.available | 2022-11-24T03:36:27Z | - |
| dc.date.copyright | 2021-08-18 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-08-03 | |
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J Cell Biochem, 121(4), 2851-2863. doi:10.1002/jcb.29519 Zhou, X., Zhang, Z., Feng, J. Q., Dusevich, V. M., Sinha, K., Zhang, H., de Crombrugghe, B. (2010). Multiple functions of Osterix are required for bone growth and homeostasis in postnatal mice. Proc Natl Acad Sci U S A, 107(29), 12919-12924. doi:10.1073/pnas.0912855107 | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/81211 | - |
| dc.description.abstract | "實驗目的 再生牙醫學是當今牙科治療的新興研究領域，此種治療主要是希望能夠借助組織工程學及再生醫學的力量修復受損的牙髓及牙周組織並保留其活性，其中，找到合適的生長因子和幹細胞是治療成功的關鍵。來自人類脫落的乳牙牙髓幹細胞(SHEDs)是容易藉由非侵入性方法取得的有效幹細胞來源，而骨形成蛋白-9(BMP-9)在目前許多研究中發現具有誘導間質幹細胞進行組織再生及分化的功能。本研究旨在通過分析相關標誌物的表現來探討BMP-9對SHEDs的生物活性的影響，標誌物包括：Osterix (Osx)、骨鈣蛋白(Osteoclacin, OCN)、鹼性磷酸酶(Alkaline phosphatase, ALP)、第一型膠原蛋白(Type I collagen)、金屬肽酶抑製劑-1(TIMP-1)、巢蛋白(Nestin)、Runx-2、神經性鈣粘附蛋白(N-cadherin)和纖溶原激活物抑制劑-1(PAI-1)等，並探討其訊號傳導路徑。 實驗方法 使用原代培養的SHEDs作為實驗細胞，並使用不同濃度的BMP-9 (0, 10, 25, 50, 100, 200 ng/ml)處理24小時後，通過即時定量聚合酶連鎖反應(Real-time quantitative PCR)、西方墨點法(western blot)以及免疫化學螢光染色法(Immunofluorescence staining)來檢測Osx、OCN、Runx-2、ALP、Type I collagen、TIMP-1、Nestin、N-cadherin和PAI-1的表現。另外，也使用western blot來檢測檢測p-Smad 1/5/8與p-Smad 2/3的蛋白表現量來研究SHEDs中BMP-9誘發的信號傳導途徑。 實驗結果： 加入BMP-9處理24小時後的SHEDs細胞顯示：Osx、OCN、ALP、Runx-2、Type I collagen、Nestin、TIMP-1、PAI-1、N-cadherin的蛋白表現量皆增加，RNA表現量Osx、OCN、Runx-2、Nestin、PAI-1、N-cadherin可以觀察到在加入BMP-9處理24小時候有些微提升，其餘組別則並沒有觀察到與控制組有明顯差異。另外也可以觀察到加入BMP-9處理24小時後，p-smad 1/5/8、p-smad 2/3以及p-p38的蛋白表現量有隨著BMP-9加入濃度上升而上升的趨勢。 結論 根據本篇研究結果可得知，BMP-9 可能具有促進SHEDs分化成骨/牙源性細胞之潛力，並具有調節SHEDs細胞外間質代謝作用。此外，Smad 1/5/8、Smad 2/3以及p-p38這三種途徑可能在加入BMP-9後的SHEDs細胞上都有被啟發進而影響SHEDs的細胞活性。SHEDs和BMP-9的共同使用未來或許可以運用在牙科組織修復工程的領域。" | zh_TW |
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| dc.description.tableofcontents | "目錄 口試委員會審定書 i 誌謝 ii 中文摘要 iii Abstract v 目錄 vii 第一章 文獻回顧 1 1.1 再生性牙科醫學(Regenerative Dentistry) 1 1.2 人類脫落乳牙牙髓幹細胞(SHEDs) 2 1.3 骨形成蛋白(Bone morphogenic protein, BMP) 3 1.3.1 BMP家族 3 1.3.2 BMP受體及傳導路徑 4 1.3.3 BMP在牙科組織再生工程的應用 5 1.3.4 BMP-9之功能及介紹 5 1.3.5 BMP-9的相關研究 7 1.4 細胞分化標誌物 8 1.4.1 Osterix (Osx, Sp7) 8 1.4.2 Runt相關轉錄因子-2 (Runx-2) 9 1.4.3 骨鈣蛋白(Osteocalcin, OCN) 9 1.4.4 鹼性磷酸酶(Alkaline phosphatase, ALP) 10 1.4.5 第一型膠原蛋白(Type I collagen, Collagen-I) 11 1.4.6 金屬肽酶抑製劑-1(TIMP-1) 11 1.4.7 纖溶原激活物抑制劑-1(PAI-1) 12 1.4.8 神經性鈣黏附蛋白(N-cadherin) 13 1.4.9 巢蛋白(Nestin) 13 第二章 實驗目的及假說 15 第三章 材料及方法 16 3.1 材料準備 16 3.2 SHEDs細胞培養 17 3.3 細胞存活率分析(MTT assay) 17 3.4 即時定量聚合酶連鎖反應(Real-time quantitative PCR) 18 3.4.1 核糖核酸(RNA)萃取 18 3.4.2 RNA定量 19 3.4.3 反轉錄(Reverse transcription) 19 3.4.4 即時定量聚合酶連鎖反應(Real-time quantitative PCR) 20 3.5 西方墨點法(Western blot) 21 3.5.1 蛋白質萃取 21 3.5.2 蛋白質定量 21 3.5.3 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(SDS-PAGE) 22 3.5.4 轉漬(Transfer gel) 23 3.5.5 封閉及抗體雜交(Blocking and antibody hybridization) 23 3.5.6 化學冷光影像擷取(Chemiluminescence photography) 23 3.6 免疫組織化學螢光染色(Immunofluorescence assay) 24 3.6.1 細胞培養 24 3.6.2 免疫組織化學螢光染色(Immunofluorescence staining) 24 第四章 實驗結果 26 4.1 BMP-9濃度對於SHED型態的影響 26 4.2 BMP-9對於SHED的影響 26 4.3 BMP-9濃度對於SHEDs蛋白質及RNA表現量的影響 26 4.3.1 BMP-9濃度對於Osterix蛋白質及RNA表現量的影響 26 4.3.2 BMP-9濃度對於Runx-2蛋白質及RNA表現量的影響 27 4.3.3 BMP-9濃度對於OCN蛋白質及RNA表現量的影響 27 4.3.4 BMP-9濃度對於ALP蛋白質及RNA表現量的影響 27 4.3.5 BMP-9濃度對於Collagen-I蛋白質及RNA表現量的影響 28 4.3.6 BMP-9濃度對於TIMP-I蛋白質及RNA表現量的影響 28 4.3.7 BMP-9濃度對於PAI-I蛋白質及RNA表現量的影響 28 4.3.8 BMP-9濃度對於N-cadherin蛋白質及RNA表現量的影響 29 4.3.9 BMP-9濃度對於Nestin蛋白質及RNA表現量的影響 29 4.4 BMP-9濃度對於磷酸化之Smad 1/5/8 、Smad 2/3及p-38蛋白質表現量的影響 30 第五章 討論 31 第六章 總結 37 參考文獻 38 表格 表格1. PCR的引子(primer)序列(sequence) 48 表格2. 西式墨點法中蛋白質萃取緩衝液(Protein extraction buffer)的調製 49 表格3. SDS-PAGE膠體的調製 49 表格4A. 西式墨點法SDS-PAGE電泳緩衝液(Running buffer)的調製 50 表格4B. 西式墨點法SDS-PAGE轉漬緩衝液(Transfer buffer)的調製 50 表格4C. 西式墨點法Tween TBS洗液的調製 51 表格5. 西式墨點法以及免疫螢光化學染色法使用的一級抗體 51 表格6. 重要名詞縮寫表 52 圖示 圖示1. SHEDs細胞在各種不同BMP-9濃度下24小時後的型態 53 圖示2. BMP-9 的濃度差異對於Osx的mRNA以及蛋白質的影響 54 圖示3. 免疫螢光化學染色的結果。BMP-9 的濃度差異對於SHEDs表現Osx的影響 55 圖示4. BMP-9 的濃度差異對於Runx-2的mRNA以及蛋白質的影響 56 圖示5. BMP-9 的濃度差異對於OCN的mRNA以及蛋白質的影響 57 圖示6. BMP-9 的濃度差異對於ALP的mRNA以及蛋白質的影響 58 圖示7. 免疫螢光化學染色的結果。BMP-9 的濃度差異對於SHEDs表現ALP的影響 59 圖示8. BMP-9 的濃度差異對於Collagen-I的mRNA以及蛋白質的影響 60 圖示9. 免疫螢光化學染色的結果。BMP-9 的濃度差異對於SHEDs表現Type-I collagen的影響 61 圖示10. BMP-9 的濃度差異對於TIMP-1的mRNA以及蛋白質的影響 62 圖示11. 免疫螢光化學染色的結果。BMP-9 的濃度差異對於SHEDs表現TIMP-1的影響 63 圖示12. BMP-9 的濃度差異對於PAI-1的mRNA以及蛋白質的影響 64 圖示13. 免疫螢光化學染色的結果。BMP-9 的濃度差異對於SHEDs表現PAI-1的影響 65 圖示14. BMP-9 的濃度差異對於N-cadherin的mRNA以及蛋白質的影響 66 圖示15. 免疫螢光化學染色的結果。BMP-9 的濃度差異對於SHEDs表現N-cadherin的影響 67 圖示16. BMP-9 的濃度差異對於Nestin的mRNA以及蛋白質的影響 68 圖示17. 免疫螢光化學染色的結果。BMP-9 的濃度差異對於SHEDs表現Nestin的影響 69 圖示18. BMP-9 的濃度差異對於p-Smad 1/5/8、p-Smad 2、p-Smad 3以及p-p38蛋白質的影響 70 圖示19. BMP-9對於SHEDs的生物活性之影響以及相關路徑 71" | |
| dc.language.iso | zh-TW | |
| dc.subject | 人類脫落乳牙牙髓幹細胞 | zh_TW |
| dc.subject | 再生牙醫學 | zh_TW |
| dc.subject | BMP-9 | zh_TW |
| dc.subject | Regenerative dentistry | en |
| dc.subject | BMP-9 | en |
| dc.subject | Stem cells from human exfoliated deciduous teeth | en |
| dc.title | BMP-9對於人類脫落乳牙牙髓幹細胞生物活性的影響暨其訊息傳導機制 | zh_TW |
| dc.title | Effect of BMP-9 on the Biological Activities of SHEDs and the Associated Signal Transduction Pathways | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張美姬(Hsin-Tsai Liu),邱賢忠(Chih-Yang Tseng),張維仁 | |
| dc.subject.keyword | 再生牙醫學,BMP-9,人類脫落乳牙牙髓幹細胞, | zh_TW |
| dc.subject.keyword | Regenerative dentistry,BMP-9,Stem cells from human exfoliated deciduous teeth, | en |
| dc.relation.page | 71 | |
| dc.identifier.doi | 10.6342/NTU202101961 | |
| dc.rights.note | 同意授權(限校園內公開) | |
| dc.date.accepted | 2021-08-04 | |
| dc.contributor.author-college | 醫學院 | 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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