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研究生:張嘉芳
研究生(外文):Chia-Fang Chang
論文名稱:Dipyridamole透過血基質氧化酵素-1抑制骨橋素表現
論文名稱(外文):Inhibition of Osteopontin Expression in NRK-52E Cells by Dipyridamole Is Mediated Through Heme Oxygenase-1
指導教授:李宏謨李宏謨引用關係
指導教授(外文):Horng-Mo Lee
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學技術學系
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:75
中文關鍵詞:骨橋素血基質氧化酵素-1訊息傳遞腎臟纖維化
外文關鍵詞:Osteopontin (OPN)DipyridamoleHeme oxygenase-1 (HO-1)Signal transductionNRK-52E tubular epithelial cellsrenal fibrosis
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骨橋素(osteopontin; OPN)是一種分泌型的磷酸化醣蛋白,在缺血性腎臟間質纖維化的過程裡扮演重要的角色。在NRK-52E大鼠腎小管上皮細胞加入化學缺氧劑氯化鈷(cobalt chloride; CoCl2),會誘導時間與劑量依存性OPN蛋白質表現增加。以l-N-acetylcysteine (l-NAC)和thenoyl-trifluoroacetone (TTFA)前處理後可阻斷CoCl2誘導OPN的增加,顯示自由基和電子傳遞複合物II可能是有關聯性的。以dipyridamole (1-10M)前處理NRK-52E細胞,可抑制CoCl2誘導OPN的表現。因為誘導血基質氧化酵素-1 (heme oxygenase-1; HO-1)的藥物已知可改善腎臟纖維化,而OPN的表現與腎臟間質纖維化有關,我們接著探討誘導HO-1的表現是否能調控OPN的表現。許多類固醇及非類固醇抗發炎藥劑均可以抑制由CoCl2所誘導的OPN表現。這些抗發炎藥劑中,rosiglitazone和dexamethasone無法誘導HO-1的表現,而celebrex和tanshinones IIA 能夠誘導HO-1的表現。以SnPP (HO-1抑制劑)前處理NRK-52E 細胞,可逆轉dipyridamole的抑制作用,顯示HO-1與dipyridamole抑制CoCl2誘導OPN的表現有關。培養細胞時加入CO釋放分子(tricarbonyldichloro-ruthenium (II)),能模擬dipyridamole抑制OPN表現的作用。以CO清除劑(血紅素;hemoglobin)前處理則能逆轉dipyridamole的抑制作用。這些資料顯示dipyridamole可能是透過誘導HO-1表現而抑制OPN的表現。增加HO-1可以催化血基質(heme)轉化出CO,進而抑制CoCl2誘導OPN的表現。我們的實驗結果顯示,HO-1的誘發劑可能適合做為腎臟纖維化的治療藥物。
Osteopontin (OPN), a secreted phospho-glycoprotein, plays a pivotal role in the progression of interstitial fibrosis in renal ischemia. In the present study, we showed that incubation of rat renal tubular NRK-52E cells with cobalt chloride (CoCl2) increased OPN protein expression in a dose- and time- dependent manner. CoCl2 induced OPN expression was blocked by pretreatment with l-N-acetylcysteine (l-NAC) and thenoyl-trifluoroacetone (TTFA) suggesting free radical production and electron transport complex II were involved. Pretreatment of cells with dipyridamole (1-10 M), inhibited the CoCl2-induced OPN expression and the inhibitory effect was associated with heme oxygenase-1 (HO-1) induction. Because induction of heme oxygenase-1 has been linked to inhibition of renal fibrosis, we tested whether induction of HO-1 modulate OPN expression in NRK-52E cells. In addition, we demonstrated that several agents that exert anti-inflammatory effects inhibited CoCl2-induced OPN expression. Among these anti-inflammatory agents, rosiglitazone and dexamethasone did not induce HO-1 expression, whereas celebrex and tanshinone IIA caused a mark HO-1 expression. Pretreatment of cells with SnPP, a HO-1 inhibitor, reversed the inhibition due to dipyridamole, suggesting that HO-1 is linked to the inhibition of CoCl2-induced OPN expression by dipyridamole. Incubation of cells with CO releasing molecule, tricarbonyldichloro-ruthenium (II) dimmer, mimicked dipyridamole’s inhibitory effect on OPN expression. Pretreatment of cells with CO scavenging agent, hemoglobin, abolished the inhibition of CoCl2-induced OPN expression by dipyridamlole. Taken together, these data suggest that dipyridamole may inhibit OPN expression through HO-1 induction. Increased HO-1 may catalyze the conversion of heme into CO, which in turn suppresses CoCl2-induced OPN expression. Our data suggest that HO-1 inducers may serve as therapeutic agents in the treatment of renal fibrosis.
中文摘要 I
Abstract III
目錄 V
圖目錄 VII
縮寫表 IX
壹、緒論 1
急性腎小管壞死(acute tubular necrosis) 2
骨橋素(Osteopontin; OPN) 2
血基質氧化酵素(Heme Oxygenases ; HOs) 5
活性氧自由基(Reactive Oxygen Species; ROS) 6
Rosiglitazone 7
Dexamethasone 8
Tanshinones 10
Celebrex (Celecoxib) 11
Dipyridamole (Persantine) 12
貳、實驗材料與方法 14
一、 實驗材料 14
1、 藥品試劑 14
2、 常用儀器 16
3、 常用溶液 18
4、 大鼠的腎臟上皮細胞 19
5、 MTT 19
7、 Rosiglitasone 20
8、 Dexamethazone 20
9、 Tanshinone IIA 20
10、Dipyridamole 20
二、實驗方法 20
1、NRK-52E cells的培養 20
2、細胞蛋白質的測定 21
3、細胞存活率試驗 22
4、流式細胞儀分析 23
5、細胞RNA萃取 25
6、聚合酵素鏈鎖反應(reverse transcriptase-polymerase chain reaction;RT-PCR) 25
7、統計分析 26
參、實驗結果 27
一、CoCl2誘導OPN的表現 27
二、ROS在CoCl2誘導OPN表現的機制中所扮演的角色 27
三、抗發炎藥物對於CoCl2誘導OPN表現的影響 29
四、Dipyridamole誘導HO-1的表現 29
五、HO-1在dipyridamole抑制CoCl2所誘導的OPN表現中所扮演的角色 30
肆、討論 32
伍、參考文獻 36
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