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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 鄭景暉(Jeng JH) | |
dc.contributor.author | Mon-Ying Lee | en |
dc.contributor.author | 李夢瑩 | zh_TW |
dc.date.accessioned | 2021-06-13T05:46:34Z | - |
dc.date.available | 2006-08-03 | |
dc.date.copyright | 2006-08-03 | |
dc.date.issued | 2006 | |
dc.date.submitted | 2006-07-13 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/33809 | - |
dc.description.abstract | 從以前的研究中發現當牙髓發炎時,細胞內的PGF2α會有上升的趨勢,然而PGF2α在人類牙髓細胞中的角色卻尚未明瞭,本篇研究的目的是利用in vitro的方法,觀察PGF2α在人類牙髓細胞中的病理生理反應,並探討PGF2α的訊息傳導路徑。我們利用反轉錄聚合酶鏈式反應(RT-PCR)和西方點墨法(Western Blotting)的方法在人類牙髓細胞中首度發現FP receptor的mRNA以及蛋白質的存在,之後我們使用西方點墨法(Western Blotting)的方法發現,當FP receptor被活化後,加入不同濃度的PGF2α(0.1, 0.5, 5, 10, 25 μM)可以誘導牙髓細胞中p38、ERK、CREB的磷酸化;再者,當我們加入不同濃度的PGF2α(0.1, 0.5, 5, 10, 25 μM)於人類牙髓細胞中培養五天後,利用Sircol Collagen Assay的方法,觀察細胞中膠原蛋白含量的表現,結果發現,加入的PGF2α會使細胞中膠原蛋白的含量下降,而且加入的PGF2α濃度越高,膠原蛋白下降的量也越多。另一方面,我們利用酵素連結免疫吸附分析(Enzyme-link immunosorbent assay,ELISA)觀察牙髓細胞在加入PGF2α後細胞內金屬蛋白酶的表現,結果發現,隨著加入的PGF2α濃度升高,細胞內total MMP-3、MMP-9會有上升的現象,而TIMP-2則有明顯的下降,這也許可以解釋加入PGF2α後,牙髓細胞內膠原內含物下降的原因。但是,我們利用MTT assay觀察同樣以不同濃度PGF2α處理的人類牙髓細胞,分析細胞的生長與存活率,結果顯示,在培養五天後,牙髓細胞的生長約受到20%的抑制,這表示PGF2α會些微的抑制牙髓細胞的生長,但是無明顯的細胞毒性反應。
另外,我們觀察PGF2α對於人類牙髓細胞中鹼性磷酸酶的影響,我們分別從細胞直接染色與細胞內mRNA的表現來觀察,利用鹼性磷酸酶染色與反轉錄聚合酶鏈式反應的方法我們發現,當以PGF2α處理人類牙髓細胞時會造成細胞中鹼性磷酸酶活性的下降,並且會使細胞內mRNA的表現降低。然而,我們進一步加入三種抑制劑:包括U0126 (a MEK inhibitor)、SQ22536 (adenylate cyclase inhibitor)、U73122(PLC inhibitor)和H89 (a PKA inhibitor)前處理並利用鹼性磷酸酶染色,結果發現,這四種抑制劑無法抑制PGF2α之作用、無法使細胞內鹼性磷酸酶的活性提昇,因此,牙髓細胞中PGF2α降低鹼性磷酸酶活性過程可能不是單獨經由ERK/CREB和PKA signaling pathways。若我們加入IL-1β(0.01、0.1、0.5、1、5μM)於牙髓細胞中,培養五天後同樣利用鹼性磷酸酶染色,發現牙髓細胞受到IL-1β的影響後,細胞內的鹼性磷酸酶活性會慢慢下降,這有可能是IL-1β誘導了牙髓細胞產生PGF2α的原因,不過確切結果還需更進一步的實驗驗證。從本實驗中我們可以發現,當人類牙髓發炎時,所產生的PGF2α會抑制牙髓細胞的生長、減低膠原蛋白,並且使細胞內鹼性磷酸酶的活性下降,有關PGF2α在人類牙髓細胞中的病理生理反應以及它對牙髓發炎之後修復所扮演的角色,仍需更進一步的研究。 | zh_TW |
dc.description.abstract | Previous studies have found that the amount of PGF2α amount in human dental pulp raise during inflammation. However the roles and effects of PGF2α in human dental pulp are not fully clear. The aim of this research was to evaluate the effects of PGF2α on human dental pulp cells in vitro. We found that primary human dental pulp cells expressed FP receptor as analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). Activation of FP receptor by PGF2α (10μM) induced both ERK and CREB phosphorylation in pulp cells as shown by western blotting. Exposure to different concentration of PGF2α (0.1, 0.5, 5, 10, 25μM) declined the collagen content of pulp cells as analyzed by Sircol Collagen Assay. PGF2α ( > 1μM) also decreased the alkaline phosphatase (ALK-P) activity and mRNA expression. However, U0126, SQ22536, U73122 and H89 (inhibitors of MEK1. adenylate cyclase, phospholipase C and protein kinase A (PKA), respectively) could not prevent the decline of ALK-P activity in dental pulp cells by exposure to PGF2α. These results indicate that PGF2α may activate FP receptors leading to ERK/CREB activation during its production in inflamed dental pulp. PGF2α attenuated the collagen turnover and ALK-P activity of pulp cells possibly via pathways not solely via ERK/CREB and adenylate cyclase/PKA activation. PGF2α is thus a crucial factor in pulpal inflammation by regulation the pulp cell behavior. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T05:46:34Z (GMT). No. of bitstreams: 1 ntu-95-R92422009-1.pdf: 1346181 bytes, checksum: f47e498cbc2dfae2b4e74e30e8065fff (MD5) Previous issue date: 2006 | en |
dc.description.tableofcontents | 第一章、前言-----------------------------------------------1
第二章、文獻回顧-------------------------------------------3 一、 前列腺素(Prostaglandins,PG)--------------------3 二、 環氧酶(cyclooxygenase ,COX)-------------------3 三、 前列腺素F2α(prostaglandin F2α,PGF2α)------------5 四、 PGF2α在牙髓生理、病理功能之研究------------------8 五、 鹼性磷酸酶(Alkaline Phosphatase,ALP)-----------9 六、 金屬蛋白水解酶(Matrix metalloproteinases,MMPs)-11 第三章、實驗動機與目的------------------------------------16 第四章、材料與方法----------------------------------------18 第五章、結果----------------------------------------------36 第六章、討論----------------------------------------------43 第七章、結論----------------------------------------------48 參考文獻-------------------------------------------------49 圖次 圖一:利用鹼性磷酸酶染色法,觀察IL-1β對於牙髓細胞鹼性磷酸酶活性的影響。(n=3)-------------------------------------70 圖二(a):加入不同濃度的IL-1β,培養五天後利用酵素連結免疫吸附分析(Enzyme-linked immunosorbent assay,ELISA)偵測牙髓細胞PGF2α的產量。(p<0.05) (n=3)-------------------------71 圖二(b):加入不同濃度的IL-1β與aspirin,培養五天後利用酵素連結免疫吸附分析(Enzyme-linked immunosorbent assay,ELISA)偵測牙髓細胞中PGF2α的含量。(p<0.05) (n=3)---------------72 圖二(c):加入不同濃度的IL-1β,培養五天後利用酵素連結免疫吸附分析(Enzyme-linked immunosorbent assay,ELISA)偵測牙髓細胞中TIMP-1的含量。(n=3)-------------------------------73 圖二(d):利用鹼性磷酸酶染色,觀察不同濃度IL-1β對牙髓細胞中鹼性磷酸酶活性的影響(上:顯微鏡下放大圖;下:肉眼觀察圖)。(n=3)----------------------------------------------------74 圖二(e):利用鹼性磷酸酶染色,觀察IL-1β與aspirin對牙髓細胞中鹼性磷酸酶活性的影響(上:肉眼觀察圖; 下:顯微鏡下放大圖)。(n=3)---------------------------------------------------75 圖三:利用鹼性磷酸酶染色,觀察PGE2對牙髓細胞中鹼性磷酸酶活性的影響(上:顯微鏡下放大圖;下:肉眼觀察圖)。(n=4)-------76 圖四(a):人類牙髓細胞中,有FP receptor mRNA的表現,由此圖中我們可以發現,人類牙髓細胞中有FP receptor的存在。(n=3)---77 圖四(b):從西方墨點法(Western Blotting)中我們發現,在人類牙髓細胞中也有FP receptor蛋白質的存在(64 KDa)。(n=3)----78 圖五:加入1.5~50μM PGF2α於牙髓細胞中,培養五天後,利用MTT assay的方法,會些微的抑制牙髓細胞的生長(約20%)。 (各組間與對照組相比,P<0.05) (n=3)-----------------------79 圖六:加入不同濃度的PGF2α,培養五天後利用Sircol Collagen Assay偵測細胞層中膠原蛋白的含量。 (各組間與對照組相比,p<0.05) (n=3)---------------------------------------------------80 圖七(a): 加入不同濃度的PGF2α,培養五天後利用酵素連結免疫吸附分析(Enzyme-linked immunosorbent assay,ELISA)偵測牙髓細胞中MMP-2的含量。(n=3)----------------------------------81 圖七(b): 加入不同濃度的PGF2α,培養五天後利用酵素連結免疫吸附分析(Enzyme-linked immunosorbent assay,ELISA)偵測牙髓細胞中MMP-3的含量。(n=3)----------------------------------82 圖七(c): 加入不同濃度的PGF2α,培養五天後利用明膠酶活性分析(Zymography)偵測牙髓細胞中MMP-9的活性。(n=2)-----------83 圖七(d): 加入不同濃度的PGF2α,培養五天後利用酵素連結免疫吸附分析(Enzyme-linked immunosorbent assay,ELISA)偵測牙髓細胞中TIMP-1的含量。(n=3)-------------------------------84 圖七(e): 加入不同濃度的PGF2α,培養五天後利用酵素連結免疫吸附分析(Enzyme-linked immunosorbent assay,ELISA)偵測牙髓細胞中TIMP-2的含量。(各組間,p<0.05) (n=3) ---------------85 圖八(a):利用鹼性磷酸酶染色與反轉錄聚合酶鏈式反應(RT-PCR)的方法發現,PGF2α能夠有效的抑制牙髓細胞鹼性磷酸酶的活性與mRNA的表現。(n=3)---------------------------------------86 圖八(b):利用鹼性磷酸酶染色,觀察不同濃度PGE2對牙髓細胞中鹼性磷酸酶活性的影響(上:顯微鏡下放大圖;下:肉眼觀察圖)。(n=4) ---------------------------------------------------------87 圖九(a):利用西方墨點法(Western Blotting)得到p38、CREB/ATF-1、ERK 1/2磷酸化表現的改變。(n=3)--------------88 圖九(b):利用西方墨點法(Western Blotting)得到COX-2訊息的表現結果。(n=3)--------------------------------------------89 圖十:利用鹼性磷酸酶染色法,觀察加入三種抑制劑(U0126、H89、SQ22536)對牙髓細胞中鹼性磷酸酶活性的影響。(n=3)--------90 表次 表一、不同濃度IL-1β對牙髓細胞PGF2α產生的影響------------92 表二、不同濃度IL-1β與aspirin對牙髓細胞PGF2α產生的影響--------------------------------------------------------94 表三、不同濃度PGF2α對牙髓細胞TIMP-2產生的影響-----------95 表四、不同濃度PGF2α對牙髓細胞細胞存活率的影響------------97 | |
dc.language.iso | zh-TW | |
dc.title | PGF2α對人類牙髓細胞病理生理功能的影響 | zh_TW |
dc.title | Pathobiological Functions of PGF2α on human dental pulp cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 94-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 王東堯(Wong TY),藍萬烘(Lan WH),張美姬(Chang MY) | |
dc.subject.keyword | 牙髓細胞,PGF2α,膠原蛋白,鹼性磷酸酶,訊息傳導, | zh_TW |
dc.subject.keyword | Dental Pulp cells,PGF2α,collagen,alkaline phosphatase (ALK-P),signal transduction, | en |
dc.relation.page | 97 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2006-07-13 | |
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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