臺灣博碩士論文加值系統

中文摘要
(Abstract in Chinese)
骨骼的主要功能是支撐我們的身體，保護體內重要器官、骨髓及維持新陳代謝和造血的功能，成熟的骨骼會以骨質重塑作用(bone remodeling cycle)來維持功能的恒定性，藉由蝕骨細胞(Osteoclast)溶解、吸收擬替換的老舊骨基質和骨母細胞(Osteoblast)不斷的構築新生骨來維持動態的生理平衡，一但骨組織受到不正常地刺激，像是cytokine、growth factors，或是endothelin-1 (ET-1) 都會影響骨母細胞的活性和增生。ET-1原是一種強效血管收縮素，後經研究證實亦具有如同TNF-和IL-1一樣的發炎調控角色，引發人體氣管發炎、皮膚炎症、腸道的克隆氏症 (Crohn’s disease)、大腸炎和肝硬化…等等發炎疾病。經研究發現，在機械性不當的刺激骨母細胞下，亦會誘發細胞產生與發炎反應相關的蛋白質COX-2和其代謝產物PGE2，而影響骨母細胞的活性。而今ET-1是否會誘發COX-2的表現來造成發炎反應及其機轉仍然未知。因此，我們擬探討在鼠骨母細胞 (Murine osteoblast-like cell line, MC3T3-E1)上，ET-1 刺激MC3T3-E1 細胞COX-2表現的分子機制為何。研究發現ET-1可經由刺激p42/44 MAPK，p38 MAPK，JNK，c-Src，PDGFR，PI3K/Akt的磷酸化和NF-B、AP-1的轉錄活性來誘發MC3T3細胞COX-2的表現，且磷酸化可被MEK1/2抑制劑(PD98059)、p38 MAPK抑制劑 (SB202190)、JNK抑制劑 (SP600125)、 Ca2+螯合劑 (BAPTA/EDTA與 TG)、 Camodulin 抑制劑 (CaMI 與KN62)、 PLC抑制劑 (D609 與 U73122)、 PKC-δ抑制劑(Rottlerin)、c-Src 抑制劑(PP1)、PDGFR 抑制劑 (AG1296)、PI3K/AKT 抑制劑 (LY294002 與 SH-5)、 NF-B 抑制劑 (Bay-117082) 和AP-1 抑制劑 (Curcumin) 所抑制。此外，當細胞進行轉染 (Transfection) MEK, ERK1, p38, JNK，PKC-δ, c-Src, p85, NIK, 和 IKK的dominant negative mutants時，都可抑制ET-1所誘發細胞COX-2的表現。Promoter luciferase assay亦證實了NF-B、AP-1及以上訊號傳遞因子對COX-2基因調控上的重要性。另外，使用 核、質分離、免疫螢光染色以及西方點漬分析法，可證實ET-1會刺激IB-的降解進而促成NF-B的核轉位(nuclear translocation) 作用，並且NF-B的核轉位作用會受到PI3K/Akt的抑制劑LY294004所抑制。同時，以ET-1 接受器ETA的拮抗劑(BQ-123)、 Gq蛋白偶聯受體拮抗劑 (GPAnt2A)、Gi蛋白偶聯受體拮抗劑 (PTX與GPAnt2) 前處理細胞皆可抑制ET-1 誘發MC3T3-E1 細胞COX-2的表現及某些磷酸激酶的磷酸化作用，經此而證實ETA、Gq蛋白和Gi蛋白的參與，並連結MAPKs與RTK轉活化之間的調節。綜合以上研究結果顯示ET-1刺激MC3T3-E1細胞COX-2的表現，會經由ETA接受器偶合Gi蛋白和PTX-sensitive Gq蛋白，MAPKs和PDGFR-PI3K/Akt轉活化的傳遞路徑，下游促成NF-B的核轉位和AP-1的活化來誘發細胞COX-2的表現。綜合以上研究結果，將可提供ET-1誘導骨母細胞COX-2表現和發炎調控的參考資料，以增加兩者之間機轉的了解，進而期許能提供治療骨骼系統致病的新方針。

Abstract
Mature bone homeostasis is maintained by bone remodeling cycle involved in osteoblast bone formation and osteoclast bone resorption. Under physiological conditions, the bone matrix is constantly renewed with bone remodeling to restructure itself in response to stimulation. Endothelin-1(ET-1), a potent vasoconstrictor, has been shown to be a pro-inflammatory cytokine like TNF- and IL-1β. ET-1 may cause a variety of inflammatory diseases, including airway inflammation, cutaneous inflammation, Crohn’s disease, ulcerative colitis, and liver cirrhosis. Under mechanical stresses, COX-2 and PGE2 expression would be induced in osteoblastic-like cells to modulate osteoblastic activities. However, the mechanisms underlying ET-1-induced COX-2 expression in osteoblastic-like cells were still unknown. Here, the mechanisms underlying ET-1-induced COX-2 expression were investigated in murine osteoblast- like cell line (MC3T3-E1). Results revealed that ET-1-induced COX-2 expression was mediated through p42/p44 MAPK, p38 MAPK, JNK, c-Src, PDGFR, PI3K/Akt phosphorylation, NF-B and AP-1 activity in MC3T3 cells. These responses were attenuated by pretreatment with the inhibitors of MEK1/2(PD98059), p38 MAPK (SB202190), JNK(SP600125), Ca2+ (BAPTA/EDTA and TG), Camodulin (CaMI and KN62), PLC (D609 and U73122), PKC-δ(Rottlerin), c-Src (PP1), PDGFR (AG1296), PI3K/AKT (LY294002 and SH-5), NF-B (Bay-117082) and AP-1 (Curcumin). The involvements of MAPKs, RTK and PI3K/AKT were also confirmed by tansfection with dominant negative mutants of MEK, ERK1, p38, JNK, PKC-δ, c-Src, p85, NIK, and IKK. Tansfection with these mutants significantly inhibited ET-1-induced COX-2 expression. Moreover, ET-1 induced transcriptional activity of a COX-2/reporter, NF-B/reporter, and AP-1/reporter constructs in MC3T3 cells. The implication of NF-kB in ET-1-mediated responses was supported by translocation of NF-kB into the nucleus and degradation of IkB-a stimulated by ET-1 with cell fraction isolation, immunofluorescence staining and Western blot. And NF-kB translocation was inhibited by LY294002. In addition, pretreatments with BQ123 (an inhibitor of ETA receptor), GPAnt2A, PTX and GPAnt2 (inhibitors of Gq and Gi-protein) attenuated ET-1-induced COX-2 expression and phosphorylation of these kinases indicating the involvements of ETA , Gq and Gi proteins, and cross linkage between MAPKs and RTK transactivation. Taken together, these results suggested that in MC3T3 osteoblast cells, ET-1 might activate a GPCR (i.e. ETA) coupling to Gq protein and Gi proteins, activating MAPKs and PDGFR-PI3K/Akt pathways, leading to NF-kB (p65) nuclear translocation and AP-1 activity to induce COX-2 expression. These results provide new insights into the mechanisms by which ET-1-induced COX-2 expression may promote inflammatory responses and will create chances for the development new therapeutic strategies for bone diseases.

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誌謝(Acknowledgement) ……………………………………………….iv
目錄(Contents)………………………………………………………...vii
縮寫表(Abbreviation) ………………………………………………viii
抑制劑表(Inhibitors) ………………………………………………. xi
中文摘要(Abstract in Chinese……………………...........xiii
英文摘要(Abstract in English)…………………………………... xv
Introduction……………………………………………..............1
Specific Aims and Significance…………………….……………..46
Materials and Methods ……………………………..............49
Results…………….……………….….........................58
Figure legends.………………………....………………..........84
Discussion………………………………………..................153
Conclusion……………………………………………………………....166
References…………………………...........................167

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