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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32873完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭景暉(Jiiang-Huei Jeng) | |
| dc.contributor.author | Tseng-Fang Tai | en |
| dc.contributor.author | 戴岑芳 | zh_TW |
| dc.date.accessioned | 2021-06-13T04:17:48Z | - |
| dc.date.available | 2006-08-03 | |
| dc.date.copyright | 2006-08-03 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-24 | |
| dc.identifier.citation | Ahn NG, Tolwinski NS, and Hughes H. U0126: An Inhibitor of MKK/ERK Signal Transduction in Mammalian Cells. Promega Notes 1999; 71: 04.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32873 | - |
| dc.description.abstract | 轉型生長因子（TGF-ßs）可能會調節人類牙髓細胞的生長、分化與功能。我們早期的研究發現TGF-ß會抑制牙髓細胞的生長及刺激膠原蛋白的合成。實驗目的：在本實驗中，我們測試了不同TGF-ß2的訊息傳導途徑對人類牙髓細胞分化所造成的影響。實驗方法：我們使用西方點墨法、免疫螢光染色、反轉錄鏈聚合酶反應(RT-PCR)、MTT測定、鹼性磷酸酶(ALP)染色與鹼性磷酸酶定量分析來觀察我們的發現。實驗結果：我們觀察到人類牙髓細胞主要表現TGF-ß1的 mRNA， TGF-ß2次之，與少量的TGF-ß3之 mRNA。而西方點墨法的結果則是發現加入TGF-ß2 (10 ng/ml)於牙髓細胞中會誘導 smad2/3，smad1/5/8與 ERK這些不同訊息傳導分子的活化;我們可用免疫螢光染色法觀察到smad3與smad1的表現從細胞質移位進入細胞核內。人類牙髓細胞經長滿五天後會表現強的鹼性磷酸酶活性，而TGF-ß2 (≧5 ng/ml)會減低牙髓細胞鹼性磷酸酶的活性(P < 0.05) 與mRNA的表現。若先將牙髓細胞以 SB431542 (TGF-ß之ALK-4, ALK-5 and ALK-7受體之抑制劑) 前處理，則結果會降低TGF-ß2 (5 ng/ml) 於細胞存活率、鹼性磷酸酶染色與鹼性磷酸酶 mRNA表現所造成的抑制作用; 此種現象在經 U0126前處理的牙髓細胞中則較不明顯。結論：從以上的實驗結果推論- TGF-ß2可能會藉著自體內泌與旁泌的作用來影響人類牙髓細胞活化不同的訊息傳導途徑。而這些不同途徑活化的結果可能會對牙髓的修復與牙本質的再生產生決定性的影響。 | zh_TW |
| dc.description.abstract | TGF-ßs (Transforming Growth Factor-ßs) may regulate the growth, differentiation and function of human dental pulp cells. Our prior study has found that TGF-ß inhibited pulp cells growth and stimulated collagen synthesis. Objectives: In this study, we tested the differential effects of TGF-ß2 signaling on differentiation of cultured human dental pulp cells. Methods: We used western blotting, immunofluorescent observation, RT-PCR, MTT assay, alkaline phosphatase (ALP) staining, alkaline phosphatase quantitative assay to test our findings. Result: We found that human dental pulp cells expressed mainly TGF-ß1, less TGF-ß2 and trace amount of TGF-ß3 mRNA. Exposure of pulp cells to TGF-ß2 (10 ng/ml) induced the phosphorylation of smad2/3, smad1/5/8 and ERK signaling pathways as analyzed by western blotting; TGF-ß2 also stimulated the nuclear translocation of smad3 and smad1 as observed by immunofluorescent microscope. Pulp cells expressed strong ALK-P activities in confluent culture for 5 days. Exposure to TGF-ß2 (≧ 5 ng/ml) decreased the ALP activity ( P < 0.05 ) and mRNA expression. Pretreatment of pulp cells with SB431542 (an inhibitor of TGF-ß ALK-4, ALK-5 and ALK-7 receptors) and to a lesser extent the U0126 (MEK1 inhibitor) prevented the inhibitory effect of TGF-ß2 (5 ng/ml) on viable cell number, ALP staining, as well as the ALP mRNA expression in dental pulp cells. Conclusion: These results suggest that TGF-ß2 may affect the biological activities of dental pulp cells via autocrine and paracrine fashion by activation the differential signal transduction pathways. These events can be crucial in pulpal repair and regeneration. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T04:17:48Z (GMT). No. of bitstreams: 1 ntu-95-R92422008-1.pdf: 12535106 bytes, checksum: 38dbaa79076f6ebcfe60ca981cc718fe (MD5) Previous issue date: 2006 | en |
| dc.description.tableofcontents | 中文摘要--------------------------------------------------------------------1
英文摘要--------------------------------------------------------------------3 第一章、文獻回顧----------------------------------------------------------------5 I. 牙髓細胞的修復-----------------------------------------------------5 II. 轉型生長因子-ß ---------------------------------------------------------7 III. TGF-ß之受體 ----------------------------------------------------------10 IV. TGF-ß 於不同細胞之訊息傳導--------------------------------------11 V. 鹼性磷酸酶-----------------------------------------------------12 VI. 鹼性磷酸酶之基因調控----------------------------------------------14 第二章、動機與目的------------------------------------------------------------17 第三章、材料與方法--------------------------------------------------------19 1.人類牙髓細胞的培養-------------------------------------------------19 2.人類牙髓細胞中TGF-ß的表現--------------------------------------19 2.1 RNA的分離方法----------------------------------------------------19 2.2 RNA定量-------------------------------------------------------------20 2.3 RNA反轉錄-----------------------------------------------------------21 2.4聚合酶鏈鎖反應----------------------------------------------------21 3. TGF-ß 在人類牙髓細胞的訊息傳導------------------------------22 3.1西方點墨法 --------------------------------------------------------22 3.2 玻片上的免疫螢光染色法 --------------------------------------24 4.牙髓細胞的生長評估-MTT assay -------------------------------25 4.1 配製MTT dye -------------------------------------------------------25 4.2 MTT assay ---------------------------------------------------------26 5.在人類牙髓細胞中鹼性磷酸酶的表現 ----------------------------26 5.1鹼性磷酸酶染色 ---------------------------------------------------26 5.2使用RT-PCR來測定鹼性磷酸酶的表現-------------------------28 5.3定量鹼性磷酸酶的活性 ------------------------------------------28 5.3.1抽取cell lysate ------------------------------------------------28 5.3.2定量DNA -----------------------------------------------------------29 5.3.3鹼性磷酸酶的活性-----------------------------------------------29 6.統計分析 ------------------------------------------------------------------30 第四章、實驗結果--------------------------------------------------------------32 1.人類牙髓細胞中TGF-ßs的表現---------------------------------------32 2. TGF-ß2在人類牙髓細胞中的訊息傳導-------------------------------32 2.1以西方點墨法分析 TGF-ß2在人類牙髓細胞的訊息傳導---32 2.2以免疫螢光染色法法分析 TGF-ß2在人類牙髓細胞的訊息傳 導---------------------------------------------------------------------------33 3.牙髓細胞的生長評估-MTT assay--------------------------------------34 4. TGF-ß2、U0126與SB431542對鹼性磷酸酶所造成的影響--------35 第五章、討論-------------------------------------------------------------39 第六章、結論-----------------------------------------------------------47 參考文獻-------------------------------------------------------------------48 表次-------------------------------------------------------------------58 表1. SDS-PAGE電泳片的配方---------------------------------------58 表2. 牙髓細胞的存活率(MTT assay)-----------------------------------59 表2~1. TGF-ß2 & U0126---------------------------------------59 表2~2. TGF-ß2 & SB431542-----------------------------------59 表2~3. 統計分析- MTT assay of U0126 & TGF-ß2----------60 表2~4. 統計分析- MTT assay of SB431542 & TGF-ß2--------61 表3. 鹼性磷酸酶定量分析- TGF-ß2------------------------------------62 表3~1. 鹼性磷酸酶定量分析----------------------------------------62 表3~2. Kruskal-Wallis Test ----------------------------------63 表3~3. Mann-Whitney Test - ALP production -------------63 表4. 鹼性磷酸酶定量分析- TGF-ß2 & U0126----------------------64 表4~1. 鹼性磷酸酶定量分析------------------------------------64 表4~2. Kruskal-Wallis Test -------------------------------65 表5. 鹼性磷酸酶定量分析- TGF-ß2 & SB431542-------66 表5~1. 鹼性磷酸酶定量分析------------------------------------66 表5~2. Kruskal-Wallis Test ----------------------------------67 表5~3. Mann-Whitney Test ----------------------------------------68 圖次-------------------------------------------------------------------69 圖1. 原生、繼生與第三代牙本質的圖示---------------------------------69 圖2. 第三代牙本質的分類-------------------------------------------------69 圖3. TGF-ß 第一型與第二型受體的結構簡圖--------------------------69 圖4. TGF-ß 之訊息傳導途徑-----------------------------------------------70 圖5. TNAP對HA 的影響-----------------------------------------------------71 圖6. 鹼性磷酸酶定量分析實驗步驟流程-------------------------------72 圖7. 人類牙髓細胞中TGF-ßs的表現-------------------------------73 圖8~1. TGF-ß2的訊息傳導途徑（不同時間點）-------------------------74 圖8~2. TGF-ß2的訊息傳導途徑（不同劑量）-------------------------75 圖8~3. 免疫螢光染色法-SMAD1-------------------------------------------76 圖8~4. 免疫螢光染色法-SMAD3-------------------------------------------77 圖9~1. MTT assay評估- TGF-ß2 & U0126---------------------------78 圖9~2. MTT assay評估- TGF-ß2 & SB431542----------------79 圖10~1. 不同劑量 TGF-ß2 之鹼性磷酸酶染色------------------------80 圖10~2. TGF-ß2 之鹼性磷酸酶產物作用之量--------------------------80 圖10~3. TGF-ß2 之DNA濃度-----------------------------------------------81 圖10~4. TGF-ß2 之鹼性磷酸酶活性--------------------------------------81 圖11~1. TGF-ß2與U0126 之鹼性磷酸酶染色--------------------------82 圖11~2. TGF-ß2與SB431542之鹼性磷酸酶染色----------------------82 圖11~3. 鹼性磷酸酶於RT-PCR的表現----------------------------------83 圖11~4. 顯微鏡下觀察鹼性磷酸酶染色結果TGF-ß2與U0126 -----83 圖11~5. 顯微鏡下觀察鹼性磷酸酶染色結果TGF-ß2與SB431542 -84 圖11~6. TGF-ß2與U0126之鹼性磷酸酶定量結果 -------------------85 圖11~7. TGF-ß2與SB431542之鹼性磷酸酶定量結果 ---------------86 | |
| dc.language.iso | zh-TW | |
| dc.subject | 轉型生長因子 | zh_TW |
| dc.subject | 鹼性磷酸酶 | zh_TW |
| dc.subject | 細胞存活率 | zh_TW |
| dc.subject | 牙髓 | zh_TW |
| dc.subject | 訊息傳導 | zh_TW |
| dc.subject | signal transduction | en |
| dc.subject | alkaline phosphatase | en |
| dc.subject | cellular survival rate | en |
| dc.subject | TGF-β | en |
| dc.subject | Pulp | en |
| dc.title | TGF- ß 2對人類牙髓細胞的影響及其訊息傳導機制 | zh_TW |
| dc.title | Effects of TGF-β2 on human dental pulp cells and its signaling | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 藍萬烘(Wan-Hong Lan) | |
| dc.contributor.oralexamcommittee | 王東堯(Tung-Yiu Wong),張美姬 | |
| dc.subject.keyword | 牙髓,轉型生長因子,細胞存活率,訊息傳導,鹼性磷酸酶, | zh_TW |
| dc.subject.keyword | Pulp,TGF-β,cellular survival rate,signal transduction,alkaline phosphatase, | en |
| dc.relation.page | 86 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2006-07-25 | |
| 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 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-95-1.pdf 未授權公開取用 | 12.24 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。