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研究生:王淑娟
研究生(外文):Shu-Chuan Wang
論文名稱:Bradykinin刺激人類纖維母細胞結締組織生長因子表現的訊息傳遞探討
論文名稱(外文):Studies on the Signaling Pathway of Bradykinin-induced CTGF Expression in Human Fibroblast Cells
指導教授:林建煌林建煌引用關係
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:中文
論文頁數:70
中文關鍵詞:人類纖維母細胞緩素MAPKAP-1
外文關鍵詞:AP-1BradykininMAPKWI-38
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Bradykinin (BK) 為kinin類的成員,是一種具有免疫活性的胜?式A為九個胺基酸所組成。 BK屬於自泌素(autacoid)的一種,在體內會誘導許多發炎的反應。結締組織生長因子(CTGF)是一種與發炎反應纖維化過程有關的蛋白。本論文所要探討的是在人類肺臟纖維母細胞中,BK誘發的CTGF蛋白表現的訊息傳導路徑。結果顯示BK隨著劑量和時間相關反應增加CTGF蛋白的表現。BK也可以依劑量增加而誘導CTGF-promoter-luciferase活性增加,且BK誘導CTGF蛋白表現可以被actinomycin D (轉錄抑制劑) 和 cycloheximide (轉譯抑制劑) 所抑制,表示BK誘導CTGF蛋白的表現是經由合成新蛋白而來。且給予B2受體拮抗劑 (HOE140) 也可以抑制BK誘導的CTGF蛋白表現;相反地,B1受體拮抗劑 (lys-(leu8)des-Arg9- bradykinin) 則不會抑制BK的反應。在人類肺纖維母細胞中,BK會使ERK、p38 MAPK和JNK三者的磷酸化。進一步給予ERK抑制劑 (PD98059)、p38 MAPK抑制劑(SB203580)和JNK抑制劑(SP600125),發現只有給予SP600125 (JNK的抑制劑) 會抑制BK誘導CTGF蛋白的表現。另外我們也使用dominant negative的JNK1及JNK2 ( JNK1 DN及JNK2 DN ) 做轉染實驗,發現JNK 1 DN 和JNK 2 DN 二者皆可抑制bradykinin誘導的CTGF蛋白的表現。此外,BK也會使c-Jun磷酸化和增加CTGF-promoter-luciferase的活性。另外使用AP-1抑制劑 (curcumin)可抑制BK誘導CTGF蛋白的表現。在Chromatin immunocipitation (ChIP) 實驗結果得知,在人類肺纖維母細胞中BK可以增加AP-1 DNA序列與蛋白的鍵結,也發現在CTGF啟動子中-747至-184的區段可增加CTGF-promoter-luciferase的活性。綜合以上的實驗結果得知,在人類肺纖維母細胞中BK經由B2受體,使JNK和c-Jun磷酸化,之後使c-Jun和c-fos與CTGF啟動子AP-1的 DNA序列結合,最終導致CTGF蛋白表現。
Bradykinin (BK), a member of kinins, is a 9 aminio acid peotide which actives the innate immune system. BK, also an autatoid, induces many inflammation response. Connective tissue growth factor (CTGF) is a one of proteins involved in fibrotic forming process in inflammatory tissues. In this study, we investigated the signaling pathway of bradykinin-induced CTGF expression in human lung fibroblast (WI-38). Bradykinin caused CTGF protein expression in concentration and time-dependent manners. Similarily, bradykinin induced an increases in CTGF-luciferase activity in a concentration-dependent manner. The bradykinin-induced CTGF expression was inhibited by the transcriptional inhibitor (actinomycin D) and the translational inhibitor (cycloheximide). The B2 receptor antagonist, HOE140, inhibited bradykinin-induced CTGF protein expression, while the B1 receptor antagonist (Lys-(leu8)des-Arg9-bradykinin) had no effect. Bradykinin activated ERK, JNK, and p38 MAPK phosphorylation. Furthermore, bradykinin-induced CTGF expression was reduced by the JNK inhibitor (SP600125), but not by the PD98059 (ERK inhibitor) and SB203580 (p38 MAPK inhibitor). The dominant negative mutants of JNK1 and JNK2 (JNK1 DN and JNK2 DN) attenuated bradykinin-induced CTGF protein expression in WI-38 cells. Bradykinin also activated c-Jun phosphorylation and induced an increase in AP-1-luciferase activity in a concentration-dependent manner. Curcumin (AP-1 inhibitor) inhibited bradykinin-induced CTGF expression in WI-38 cells. Bradykinin causes increases in the formation of an AP-1-specific DNA protein complex by ChIP assay. Bradykinin-induced CTGF-luciferase was controlled by the sequence -747 to -184 bp upstream of the transcription start site on the human CTGF promoter. In conclusion, bradykinin activated JNK to induce c-Jun and AP-1 activation, which in turn induced CTGF expression in WI-38 fibroblast cells.
壹、 中文摘要…………………………………………………1-2
貳、 英文摘要…………………………………………………3-4
參、 緒論………………………………………………………5-11
肆、 實驗材料與方法………………………………………...12-16
一、 實驗材料…………………………….……………...12-13
二、 實驗方法……………………………………………13-16
伍、 結果………………………………………....…………....17-23
一、 BK 刺激WI-38細胞CTGF蛋白的表現......................17
二、 BK 刺激WI-38細胞CTGF-luciferase活性增加.........17
三、 BK誘導CTGF蛋白表現是經由合成新蛋白而來........18
四、 BK是經由B2受體刺激CTGF蛋白的表現.................18
五、 BK引發ERK1/2, p38 MAPK及JNK的磷酸化...........19
六、 BK經由JNK調控CTGF蛋白的表現.......................19-20
七、 JNK1 DN 及JNK2 DN抑制BK誘導的CTGF蛋白的表現.......................................................................................20
八、 BK引發c-Jun磷酸化和AP-1參與CTGF蛋白的表現...................................................................................20-21
九、 BK透過CTGF啟動子中-747到-184位置誘導CTGF蛋白表現..............................................................................21-22
十、 BK誘導AP-1的活化.................................................22-23
陸、 討論.....................................................................................24-28
柒、 文獻參考............................................................................ 29-36
捌、 圖表.....................................................................................37-57
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