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研究生:王禎瑩
研究生(外文):Jen-Ying Wang
論文名稱:腺苷磷酸活化激酶於人類支氣管上皮細胞因木屑煙萃取物誘導白細胞介素-8基因表現所扮演的角色
論文名稱(外文):Role of AMP-activated protein kinase in the Up-regulation of IL-8 Induced by Wood Smoke Extract in Human Bronchial Epithelial Cells
指導教授:高毓儒
指導教授(外文):Yu Ru Kou
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生理學研究所
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:2009
畢業學年度:97
語文別:中文
論文頁數:89
中文關鍵詞:腺苷磷酸活化激酶木屑煙萃取物白細胞介素-8
外文關鍵詞:AMP-activated protein kinaseWood Smoke ExtractIL-8
相關次數:
  • 被引用被引用:1
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  • 下載下載:27
  • 收藏至我的研究室書目清單書目收藏:0
毒煙的吸入會導致肺部發炎甚至對生命造成威脅,因此當肺部上皮細胞 (lung epithelial cells) 受到刺激物或有毒物質傷害時,會促進各種不同趨化激素 (chemokines) 產生,包括白細胞介素-8 (interleukin-8, IL-8),IL-8是一種前趨炎物質 (proinflammatory),使嗜中性白血球活化並且吸引嗜中性白血球由血液循環中滲透到受損的組織部位而引起發炎反應,所以肺部上皮細胞在肺部發炎的驅動與進展扮演一個重要的角色。根據文獻指出當肺部上皮細胞受到有害物質刺激時,會活化上皮細胞內的有絲分裂活化蛋白質激酶 (mitogen-activated protein kinases, MAPKs) 訊息傳遞路徑,並且需要藉由活化轉錄因子 (transcription factor),如訊息轉導與轉錄活化因子 (signal transducers and activators of transcription protein, STAT) 與核轉錄因子-kappa B (nuclear factor-kappa B) 導致IL-8表現增加,然而,這些發炎性刺激物造成IL-8表現增加的整個機制仍然不清楚。此外,已知腺苷磷酸活化激酶 (AMP-activated protein kinase, AMPK) 是一種絲氨酸/蘇氨酸蛋白激酶 (serine/threonine kinase) 與調控能量的代謝有關,然而,有研究指出在滑液的纖維母細胞,當AMPK活化後經由p38/NF-�羠訊息傳遞路徑導致發炎反應產生;相對地,也有文獻指出AMPK磷酸化會抑制脂多醣 (lipopolysaccharide, LPS) 刺激細胞所產生的發炎反應,因此,本實驗主要探討:1) 在人類支氣管上皮細胞中AMPK是否參與調控木屑煙 (wood smake extract, SE) 所誘導IL-8表現,2) 以及是透過哪些訊息傳遞路徑進而調控IL-8表現。由實驗結果得知,發現給予細胞SE刺激,IL-8蛋白製造的程度上會隨著SE濃度增加及SE作用時間加長而上升,SE在濃度25 μg/ml或24小時的組別,IL-8蛋白的表現最高,在50、100 μg/ml這兩組IL-8蛋白的表現逐漸減少,在SE刺激細胞後,AMPK於30分鐘後活化,利用AMPK抑制劑compound C,可降低IL-8蛋白的表現;預先給予細胞處理轉錄因子或者MAPKs的抑制劑,再給予SE刺激,得知SE刺激細胞造成IL-8蛋白增加是透過轉錄因子NF-�羠與STAT以及胞外信號調節激酶 (extraceullar signal-regulated kinase, ERK) 與c-Jun N端蛋白質激酶 (c-Jun N-terminal kinase, JNK),利用AMPK抑制劑compound C,可以減少轉錄因子NF-�羠與STAT進入細胞核以及ERK與JNK活化現象。另外,預先給予細胞處理MAPKs的抑制劑,也可以減少轉錄因子NF-�羠與STAT進入細胞核,綜合以上實驗結果得知:1) SE 25 μl/ml刺激支氣管上皮細胞透過AMPK活化導致IL-8表現增加,利用AMPK抑制劑compound C,可降低IL-8蛋白的表現;2) AMPK被活化後,進而活化MAPKs中的ERK與JNK,促使轉錄因子STAT3及NF-kappa�羠活化,造成IL-8表現增加。
Inhalation of toxic smoke causes severe pulmonary inflammation, which is life-threatening. Because their accessibility to toxic smoke, lung epithelial cells (LECs) play a critical role in the initiation and progression of this pulmonary inflammation. Direct exposure of LECs to inflammatory stimuli other than toxic smoke has been shown to induce increases in the production of various chemokines including interleukin-8 (IL-8). Indeed, IL-8 has been recognized as the key chemokine for trafficking of inflammatory cells to the alveolar spaces. In LECs, the up-regulation of IL-8 induced by these stimuli involves signaling pathways such as mitogen-activated protein kinases (MAPKs) and requires transcriptional factors such as signal transducers and activators of transcription protein (STAT) and nuclear factor-kappa B signaling pathway. Whether toxic smoke can stimulate LECs leading to up-regulation of IL-8 remains unclear. To this end, AMP-activated protein kinase (AMPK), a serine/threonine kinase possessing energy-sensing capability, is well known for its function in the activation of ATP-generating pathway. However, recent evidence suggests that AMPK may functionally modulate acute inflammatory responses in cell type other than LECs. For example, activation of AMPK increases the production of inflammatory cytokines via p38/NF-�羠 signaling pathway in human synovial fibroblast. Contrastingly, activation of AMPK attenuates lipopolysaccharide-induced neutrophil pro-inflammatory activity. Accordingly, the objectives of this study were to investigate 1) whether exposure of cultured human bronchial epithelial cells (HBECs) to wood smoke extract (SE) may up-regulate IL-8 and, if so, 2) the signaling pathways responsible for this IL-8 induction. We found that SE exposure increased protein expression of IL-8 in a dose- and time-dependant manner. Studies using specific pharmacological inhibitors revealed that the SE-induced up-regulation of IL-8 was mediated through AMPK, ERK (one major member of MAPKs) and JNK (one major member of MAPKs) signaling pathways, and depended on transcriptional factors NF-kappa B and STAT. Further investigations indicated that SE exposure increased phosphorylation of AMPK and promoted the NF-kappa B and STAT3 translocation to nucleus, but not STAT5, and Ikappa B-alpha degradation in the cytosol. Pharmacological inhibition of AMPK resulted in a reduction of SE-induced activation of NF-kappa B and STAT3. Additionally, in presence of PD98059 or SP600125 (a specific inhibitor of ERK or JNK), SE-induced translocation of NF-kappa B and STAT3 were reduced. Furthermore, the SE-induced activation of ERK and JNK was mediated through AMPK phosphorylation. These results suggest that SE-induced up-regulation of IL-8 is mediated through AMPK activation, which leads to NF-kappa B and STAT3 translocation to nucleus via ERK and JNK phosphprylation
目錄 i
圖目錄 iii
縮寫對照表 iv
中文摘要 vi
Abstract viii
壹、 文獻回顧及研究目的 1
一、 火災煙的毒性: 2
二、 火災煙造成肺部損傷 4
三、 呼吸道上皮細胞 (Airway epithelial cells) 5
四、 白細胞介素-8 (Interleukin-8, IL-8) 6
五、 火災煙造成的發炎反應 7
六、 腺苷單磷酸活化激酶 (AMP-activated protein kinase, AMPK) 13
七、 研究目的 15
貳、 材料與方法 17
一、 儀器設備: 18
二、 實驗材料與試劑: 19
三、 實驗用溶液配方: 21
四、 實驗方法: 24
參、 實驗結果 36
一、 SE刺激支氣管上皮細胞對於IL-8之影響 37
二、 SE對於AMPK activation之影響 38
三、 AMPK activation對於SE誘導細胞IL-8表現之影響 39
四、 Transcription factors inhibitors對於SE誘導細胞IL-8表現之影響 40
五、 SE對於細胞內NF-kappa B訊號傳遞路徑的影響 41
六、 Compound C抑制AMPK activation對於SE調控細胞內NF-kappa B之p65進入細胞核之影響 41
七、 SE對於細胞內STAT5之影響 42
八、 Compound C抑制AMPK activation對於SE調控細胞內STAT3進入細胞核之影響 43
九、 MAPK inhibitors對於SE誘導細胞IL-8表現之影響 44
十、 MAPK inhibitors對於SE調控細胞內NF-kappa B之p65與STAT3之進入細胞核之影響 46
十一、 Compound C抑制AMPK activation對於SE調控細胞內ERK activation之影響 47
十二、 Compound C抑制AMPK activation對於SE調控細胞內JNK activation之影響 48
肆、 討論與結論 49
一、 SE誘導人類支氣管上皮細胞IL-8蛋白製造增加 50
二、 AMPK activation調控SE誘導細胞IL-8蛋白表現 51
三、 SE刺激細胞透過AMPK activation造成轉錄因子NF-kappa B與STAT調控IL-8蛋白表現 52
四、 SE刺激細胞透過AMPK activation活化ERK與JNK造成轉錄因子NF-kappa B與STAT調控IL-8蛋白表現 53
伍、 附圖 55
陸、 參考文獻 68
附件一 77
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