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研究生:鄭琇丹
研究生(外文):Hsiu-Tan Cheng
論文名稱:乙型轉型生長因子藉由多重路徑抑制人類初代滑膜纖維母細胞上第一型間白素誘導之第二型蛋白酶活化受體表現
論文名稱(外文):TGF-b inhibits PAR-2 expression induced by IL-1b on human primary synovial fibroblasts through multiple pathways
指導教授:陳建和陳建和引用關係
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學科學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:英文
論文頁數:73
中文關鍵詞:骨關節炎第二型蛋白酶活化受體第一型間白素乙型轉型生長因子
外文關鍵詞:OAPAR-2IL-1bTGF-b
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蛋白酶活化受體家族屬於GPCR,由四個成員組成,分別是第一型到第四型蛋白酶活化受體,四個成員之中,以第二型和發炎反應最為相關。目前已知第二型蛋白酶活化受體在關節炎病人以及正常人的軟骨細胞均有,其中在關節炎病人的軟骨細胞上表現較正常人多,並且其表現會受第一型間白素影響而上升,但是此上升現象在乙型轉型生長因子存在下則會被抑制,而乙型轉型生長因子在關節炎中是重要的同化作用因子。這些發現表示第二型蛋白酶活化受體和骨關節炎病程機制有所關聯。雖然已經確定乙型轉型生長因子會抑制關節炎病人軟骨細胞上的第二型蛋白酶活化受體表現,但其詳細分子機制仍然未知,並且,這些現象在人類滑膜纖維母細胞模式中,並未被研究過,因此,本篇研究即是探討人類滑膜纖維母細胞模式中,乙型轉型生長因子如何抑制第二型蛋白酶活化受體表現。我們在本研究中已經證實了,在人類滑膜纖維母細胞中,第一型間白素會經由p38 路徑引發第二型蛋白酶活化受體表現,並且此種誘導也可被乙型轉型生長因子抑制。同時,因為此種抑制作用可持續達四十八小時,因此,我們認為乙型轉型生長因子對第二型蛋白酶活化受體的抑制行為可能是受到多條路徑所調控。首先,我們發現乙型轉型生長因子會在短時間內直接抑制p38路徑;其次,我們也證實了此種抑制現象間接和MMP-13的去活化有關;最後,乙型轉型生長因子誘導結締組織生長因子產生後,p38路徑也會被結締組織生長因子所抑制。在人類滑膜纖維母細胞中,當第一型間白素存在時,第二型蛋白酶活化受體基因表現在很短時間內即被活化,此表示第二型蛋白酶活化受體在骨關節炎病程早期佔有重要角色,有鑑於此,抑制第二型蛋白酶活化受體在關節炎初期的表現,是避免關節炎病程進展的理想方式。
Proteinase-activated receptors (PARs) are a family of four G-protein-coupled receptors (GPCR) which included four members: PAR-1, PAR-2, PAR-3, and PAR-4. As reported, PAR-2 related mostly with inflammation. Osteoarthritis (OA) patients, chondrocytes have higher PAR-2 expression level than that of normal chondrocytes and this expression can be up-regulated by IL-1b but repressed by TGF-b, an anabolic factor of OA pathogenesis. This suggests the relationship between PAR-2 and OA progression. Even though it is known that TGF-b can inhibit PAR-2 expression in OA chondrocytes, the phenomenon and the mechanisms have never been discussed in human primary synovial cells. The aim of this study is to investigate the mechanism how TGF-b represses IL-1b induced PAR-2 expression in human primary synovial cells. In this study, we have demonstrated that IL-1b induces PAR-2 expression via p38 pathway and this induction can be repressed by TGF-b in human primary synovial cells. Also, we observed that this inhibition persists for at least 48 hours, and this suggests TGF-b inhibits PAR-2 expression through multiple pathways. First, TGF-b inhibits PAR-2 by inhibiting IL-1b induced p38 signal transduction. Then, we demonstrated that the inhibition also indirectly due to MMP-13 inactivation. Finally, TGF-b induces CTGF, and CTGF also represses PAR-2 expression by inhibit IL-1b induced phospho-p38 level. Because PAR-2 gene expression is up-regulated by IL-1b in short time in human primary synovial cells, this suggests that PAR-2 expression may play an important role in early phase of OA. In light of this, PAR-2 might be a novel ideal therapeutic target to prevent OA from progressing.
致謝................................................................................................................... I
Abstract............................................................................................................. II
摘要.................................................................................................................... III
Table of content...................................... IV
Introduction....................................................................................................... 1
Materials and Methods.................................................................................... 6
Materials......................................................................................................... 6
Primary culture of human synovial cells........................................................ 7
RNA extraction and RT-PCR......................................................................... 8
Polymerase Chain Reaction............................................................................ 9
Preparation of cell lysates............................................................................... 10
SDS PAGE……………….............................................................................. 10
Western Blotting............................................................................................. 10
Preparation of medium protein……............................................................... 11
Collagen zymography………......................................................................... 11
Statistical analysis.......................................................................................... 11
Results................................................................................................................ 13
Induction of PAR-2 mRNA and protein level by IL-1b in human primary synovial cells.................................................................................................. 13
Inhibition of IL-1b -induced PAR-2 expression by p38 MAPK inhibitor but not by MEK or JNK MAPK inhititor in human primary synovial cells... 13
TGF-b1 suppresses excess expression PAR-2 level induced by IL-1b in human primary synovial cells......................................................................... 14
TGF-b1 inhibits IL-1b mediated p38 MAPK activation, but not JNK or ERK level of human primary synovial cells................................................... 14
TGF-b1 induces CTGF mRNA and protein expression level of human primary synovial cells..................................................................................... 15
CTGF suppresses PAR-2 protein level induced by IL-1b in human primary synovial cells..................................................................................... 15
CTGF inhibits IL-1b-mediated p38 MAPK activation, but activated ERK pathway of human primary synovial cells.............................................. 16
TGF-b1 down-regulates TIMP-3 mRNA and protein level of human primary synovial cells..................................................................................... 17
TGF-b1 induces TIMP-3 expression via Akt pathway in human primary synovial cells.................................................................................................. 17
TIMP-3 blocks MMP-13 activity in culture medium of human primary synovial cells................................................................................................... 18
Blocking of MMP-13 function resulted in down-regulation of PAR-2 protein level of human primary synovial cells............................................... 18
Discussion.......................................................................................................... 19
References......................................................................................................... 22
Figures............................................................................................................... 28
Figure 1........................................................................................................... 28
Figure 2........................................................................................................... 31
Figure 3........................................................................................................... 33
Figure 4........................................................................................................... 36
Figure 5........................................................................................................... 39
Figure 6........................................................................................................... 43
Figure 7........................................................................................................... 45
Figure 8........................................................................................................... 53
Figure 9........................................................................................................... 56
Figure 10......................................................................................................... 60
Figure 11......................................................................................................... 62
Appendix Figures.............................................................................................. 64
AF. 1............................................................................................................... 65
AF. 2............................................................................................................... 66
AF. 3............................................................................................................... 69
AF. 4............................................................................................................... 70
AF. 5............................................................................................................... 71
AF. 6............................................................................................................... 72
AF. 7............................................................................................................... 73
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