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研究生(中)蘇德軒
研究生(英)Su De Shiuan
論文名稱(中)評估亞美罌粟鹼對於脂多醣在大鼠腎間膈細胞中所誘導發炎性細胞激素表現之抑制效果
論文名稱(英)Evaluation of Armepavine Inhibiting LPS-Induced Inflammatory Cytokines Expression in Rat Mesangial Cells
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指導教授(中)郭育綺
指導教授(英)Kuo Yuh Chi
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畢業學年度94
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語文別中文
關鍵字(中)亞美罌粟鹼 細胞激素 腎絲球腎炎 脂多醣 腎間膈細胞
關鍵字(英)armepavine TNF-α IL-6 mesangial cells Lipopolysaccharide
摘要(中)根據行政院衛生署近十年的統計,發現每年死於腎炎(Nephritis) 的人數節節上升,為國人十大死因之ㄧ,因此值得我們進行深入探討與研究。腎絲球腎炎(Glomerulonephritis;GN)為腎炎中發生率極高的類型之ㄧ。雖然其真正發病原因 並不明瞭,但推測可能原因包括有,腎絲球中沉積大量的免疫複合體(Immunocomplex)、腎間膈細胞(Mesangial cell)的異常增生、發炎性細胞激素(Inflamatory cytokines)的過度產生。然而,部份疾病也極易併發腎絲球腎炎,例如糖尿病、高血壓、紅瘡性狼斑、上呼吸道感染等。根據許多文獻報導得知,腎間膈細胞的過度增生與否,已成為腎絲球腎炎的重要指標之ㄧ。脂多醣(Lipopolysaccharide;LPS)能夠刺激腎間膈細胞釋放M-CSF與ICAM-1,以及多種發炎性細胞激素,包括IL-1β、IL-6、TNF-α等。由本實驗室過去的研究結果發現,由蓮子(Nelumbo nucifera)純化之有效成分,亞美罌粟鹼(Armepavine; C19H23O3N;MW313)具有良好之抗發炎與免疫調控之活性,因此在本研究中,探討亞美罌粟鹼對於在腎間膈細胞中,LPS所誘導發炎性細胞激素基因表現之影響,以評估亞美罌粟鹼未來減緩腎炎疾病之潛力。由結果發現,LPS確實能引發大鼠腎間膈細胞產生IL-6以及TNF-α等發炎性細胞激素,而亞美罌粟鹼可以明顯抑制IL-6以及TNF-α之表現,並呈現劑量性反應。利用各種訊息抑制劑加入,證明LPS引起TNF-α基因於大鼠腎間膈細胞中表現與Mitogen-activated protein kinase (MAPK)、NF-κB與Phosphoinositide 3-kinase (PI3K)等訊息傳導途徑有關。經實驗證實亞美罌粟鹼對於LPS所引起之MAPK與NF-κB活化具有明顯抑制作用,而在高濃度情況下,對於PI3K活性亦具有抑制效果。因此推測,亞美罌粟鹼可以藉由調控MAPK、NF-κB與PI3K之活化,而降低TNF-α基因於大鼠腎間膈細胞中之表現。
摘要(英)According to the information from Department of Health, Executive Yuan, Taiwan, people died in nephropathy gradually increased. Glomerulonephritis is the most occurrence disease in all kinds of nephritis. Many studies indicate the pathogenesis of glomerulonephritis is related to immune complexes deposition, mesangial cells proliferation, and inflammatory cytokines overexpression such as interleukin-1β (IL-β), IL-6, and tumor necrosis factor-α (TNF-α). Moreover, some diseases including diabetes, hypertension, systemic lupus erythematosus, and upper respiratory tract infection often accompany glomerulonephritis. Previous studies indicate mesangial cells proliferation is one of the diagnostic markers of glomerulonephritis. Lipopolysaccharide (LPS) stimulated mesangial cells to release numerous inflammatory cytokines, such as IL-1β, IL-6, and TNF-α. They also secrete M-CSF and ICAM-1 to recruit macrophages. Our previous studies indicate that armepavine (C19H23O3N; MW313) purified from Nelumbo nucifera possesses immune modulatory functions. To evaluate the healing effects of armepavine on nephritis, the rat mesangial cells were applied as target cells, and effects of armepavine on inflammatory cytokines expression such as TNF-α and IL-6 were determined. The results demonstrated that LPS stimulated IL-6 and TNF-α mRNA expression in rat mesangial cells. However, armepavine decreased the levels of these transcripts in the cells with a dose-dependent manner. The results demonstrated that LPS stimulated TNF-α gene expression in rat mesangial cells by activation of mitogen-activated protein kinase (MAPK), NF-κB, and phosphoinositide 3-kinase (PI3K) pathways. The data proved that armepavine inhibited activation of MAPK, PI3K, and NF-κB in rat mesangial cells induced by LPS. Thus, we suggested that armepavine decreased TNF-α gene expression in rat mesangial cells through modulation of MAPK, NF-κB and PI3K activation.
論文目次目錄 頁 中英縮寫對照表 1 中文摘要 3 英文摘要 4 壹、序論 5 一、亞美罌粟鹼(Armepavine)之簡介 5 二、腎臟之結構與功能 6 三、腎炎(Nephritis)之分類與成因 6 四、腎絲球腎炎中細胞激素之致病角色 7 五、腎絲球腎炎之治療 9 六、腎間膈細胞 9 七、脂多醣引發之細胞訊息 10 八、研究動機與目的 11 貳、材料與方法 13 一、亞美罌粟鹼之合成 13 二、細胞株之培養 13 三、細胞存活率測試 14 四、全量細胞核醣核酸之製備 14 五、反轉錄聚合?連鎖反應 15 六、蛋白質之製備 16 七、蛋白質定量 17 八、?酸十二酯鈉-聚丙烯銨膠體電泳法 17 九、西方墨點分析 18 十、共軛焦顯微鏡分析 18 ?、實驗結果 20 一、LPS誘導大鼠腎間膈細胞表現TNF-α與IL-6之 mRNA 20 二、亞美罌粟鹼降低LPS誘導大鼠腎間膈細胞中TNF-α mRNA表現之影響 20 三、亞美罌粟鹼對於LPS誘導大鼠腎間膈細胞中IL-6 mRNA表現之影響 21 四、亞美罌粟鹼對於大鼠腎間膈細胞存活率之影響 22 五、不同訊息傳導抑制劑對於LPS誘導大鼠腎間膈細 胞中TNF-α mRNA表現之影響 23 六、亞美罌粟鹼對於LPS誘導大鼠腎間膈細胞中ERK 路徑之影響 24 七、亞美罌粟鹼對於LPS誘導大鼠腎間膈細胞中p38 路徑之影響 25 八、亞美罌粟鹼對於LPS誘導大鼠腎間膈細胞中JNK 路徑之影響 26 九、亞美罌粟鹼對於LPS誘導大鼠腎間膈細胞中NF-κB 路徑之影響 26 十、亞美罌粟鹼對於LPS誘導大鼠腎間膈細胞中PI3K 路徑之影響 27 十一、亞美罌粟鹼對於LPS誘導未同步化大鼠腎間膈 細胞中TNF-α與IL-6 mRNA表現之影響 28 十二、亞美罌粟鹼與U0126對於LPS誘導大鼠腎間膈 細胞中TNF-α與IL-6 mRNA表現之影響 29 肆、討論 31 伍、參考資料 37 表 43 表一:行政院衛生署之腎臟疾病死亡人數 43 表二:大鼠腎間膈細胞中基因表現之引子序列 44 表三:聚合??鎖反應條件 45 圖 46 圖一:亞美罌粟鹼之結構式 46 圖二:脂多醣引發之細胞訊息 47 圖三:LPS對於大鼠腎間膈細胞中發炎性細胞激素 mRNA表現之影響 48 圖四:亞美罌粟鹼對於LPS誘導腎間膈細胞中TNF-α mRNA表現之影響 49 圖五:亞美罌粟鹼對於LPS誘導腎間膈細胞中IL-6 mRNA表現之影響 50 圖六:亞美罌粟鹼對於大鼠腎間膈細胞存活率之影響 51 圖七:不同訊息傳導抑制劑對於LPS誘導大鼠腎間膈 細胞中TNF-α mRNA表現之影響 52 圖八:亞美罌粟鹼對於LPS所誘導大鼠腎間膈細胞中 ERK磷酸化之影響 53 圖九:亞美罌粟鹼對於LPS所誘導大鼠腎間膈細胞中 p38磷酸化之影響 54 圖十:亞美罌粟鹼對於LPS所誘導大鼠腎間膈細胞中 JNK磷酸化之影響 55 圖十一:亞美罌粟鹼對於LPS誘導大鼠腎間膈細胞中 NF-κB轉位之影響 56 圖十二:亞美罌粟鹼對於LPS所誘導大鼠腎間膈細胞中 AKT磷酸化之影響 57 圖十三:亞美罌粟鹼對於LPS誘導未同步化大鼠腎間膈 細胞中TNF-α與IL-6 mRNA表現之影響 58 圖十四:亞美罌粟鹼與U0126對於LPS誘導大鼠腎間膈 細胞中TNF-α與IL-6 mRNA表現之影響 59
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