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| 題名: | 腎素-血管張力素系統調控在高氧環境下膠原蛋白過度表現之機制探討 |
| 作者: | 藍耀東 |
| 貢獻者: | 醫學科學研究所 |
| 日期: | 2010 |
| 上傳時間: | 2010-08-31 10:57:16 (UTC+8) |
| 摘要: | 肺臟支氣管發育不全(bronchopulmonary dysplasia, BPD)是新生兒在出生時因呼吸困難所使用高濃度氧氣和正壓呼吸器後所產生的一種慢性肺臟疾病。BPD病人的肺泡發育遲緩，且肺纖維化程度增加，導致少年和青少年的罹病率和死亡率增加。研究發現新生鼠長期暴露於 hyperoxia 的環境中，也有肺泡生成和微血管發育受損及增加肺臟纖維化的現象，與BPD的病理特徵相似；因此推測hyperoxia為BPD的一個危險因子。雖然在新型BPD中，fibroproliferation並不明顯，但是hyperoxia仍會造成急性肺臟損傷和導致肺臟纖維化；然而，目前並無有效的方法治療BPD。因此，研究新型BPD產生的機制是很重要的。
腎素-血管收縮素系統(renin-angiotensin system, RAS)是調控血壓和體液恆定的一個關鍵因子。血管收縮素II (angiotensin II, Ang II)是RAS的主要調控分子，它是由血管收縮素原(angiotensiongen, AGT)被腎素(renin)及血管收縮素轉化酶(angiotensin I converting enzyme, ACE)作用而產生的。RAS除了可作用於全身組織外，也可以在局部器官產生而僅影響器官內的反應。例如，Ang II具有自體分泌(autocrine)，對位分泌(paracrine)的生理活性進而改變肺臟器官內的細胞生理作用。文獻指出Ang II可引發肺臟纖維母細胞的增生及刺激其產生膠原蛋白；但是hyperoxia誘導肺纖維化的機制是否經由RAS的代謝途徑尚未釐清，而Ang II在Ang II是否可以做為治療 hyperoxia 所誘發肺纖維化的藥物標的物仍是未知的。
本論文利用人類胚胎肺臟纖維母細胞(MRC-5 cells)和新生鼠暴露在hyperoxia的環境下，研究RAS的代謝途徑與膠原蛋白的生合成間的關係。飼養新生鼠在大於95%的氧氣一週後，再暴露大於60%的氧氣二週。實驗結果顯示，hyperoxia 可顯著的增加新生鼠肺部總可溶性膠原蛋白量、第一型膠原蛋白和α-smooth muscle actin (α-SMA)的基因和蛋白表現。RAS中的各相關分子包括AGT、ACE和Ang II的生成也隨著hyperoxia刺激後而顯著增加。Hyperoxia也引發Ang II type I receptor (AT1R)的基因和蛋白表現卻沒有顯著改變AT2R的表現。進一步研究發現，若以AT1R small interfering RNA減少MRC-5 產生AT1R，可有效抑制hyperoxia所誘使的phosphorylated-ERK (p-ERK)、α-SMA和第一型膠原蛋白表現。此外，若細胞預先處理Ang II peptide、同時投與AT1R抑制劑losartan或AT2R抑制劑PD123319；我們觀察到無論在normoxia或是hyperoxia狀態下，Ang II總是增加p-ERK和α-SMA和第一型膠原蛋白表現，此種效果可被losartan抑制，但不受PD123319的影響。而在動物實驗結果顯示，相較於room air組，hyperoxia組中僅僅只有ERK的訊息傳遞被活化，而p38 mitogen-activated protein kinase (MAPK)和c-Jun N-terminal kinase (JNK)並沒有被影響。總結以上結果推論在MRC-5細胞和新生鼠中，hyperoxia誘發膠原蛋白生合成會經由RAS的活化，而ERK的磷酸化可能參與在RAS的訊息傳遞路徑中。
Bronchopulmonary dysplasia (BPD) is a chronic lung disease that develops in newborn infants treated with oxygen and positive pressure ventilation for respiratory distress at birth. BPD is characterized by decreased alveolar and capillary development and increased fibrosis and is a major cause of morbidity and mortality throughout childhood and young adulthood. Prolonged exposure of neonatal mice to hyperoxia results in impaired alveolarization and capillary development and increases lung fibrosis that is very similar to human BPD. In addition, hyperoxia is identified as a risk factor that contributes to the development of BPD. Although fibroproliferation is less prominent in new BPD, hyperoxia still cause acute lung injury and lead to pulmonary fibrosis. However, no effective therapy is established for treatment of BPD. Therefore, it is important to explore new strategies for the BPD treatment.
The renin-angiotensin system (RAS) is a key regulator of blood pressure and fluid homeostasis. Angiotensin (Ang) II is main effector molecule of the RAS and is produced from the substrate angiotensinogen (AGT) through sequential enzymatic cleavages by renin and angiotensin I converting enzyme (ACE). The compartmentalized RAS may work within individual organ system with some degree of autonomy to influence regional response. In addition, Ang II may have autocrine and paracrine actions at the cellular level in the lung tissue. It has been reported that Ang II induces human lung fibroblast proliferation and stimulates collagen synthesis. However, the relationship of RAS in the pathogenesis of hyperoxia-induced lung fibrosis has not yet been established. In addition, the role of Ang II in the pathophysiology of hyperoxia-induced pulmonary fibrosis and the therapeutic potential for targeting Ang II in pulmonary fibrosis is still unclear.
The aims of this study were to investigate the effects of hyperoxia on the components of the RAS and collagen expression in human lung fibroblasts (MRC-5) as a cell model and in newborn rats as an animal model. Rat pups were exposed to 1 week of > 95% O2 and further 2 weeks of > 60% O2. Hyperoxia significantly increased total collagen, collagen type I, and α-smooth muscle actin (α-SMA) mRNA and protein expression in vitro and in vivo. RAS components including angiotensinogen (AGT), the ACE and Ang II production were also significantly upregulated after hyperoxic stimulus. Hyperoxia also induced Ang II type I receptor (AT1R) expression but did not alter AT2R expression in cell lines and rat lungs. Furthermore, silencing of AT1R signaling with small interfering RNA suppressed hyperoxia-induced the phosphorylated extracellular signal-regulated kinase (p-ERK) 1/2, α-SMA, and collagen type I expression. Moreover, Ang II increased p-ERK 1/2 and collagen type I expression, and these increases were inhibited by the AT1R inhibitor, losartan, but not by the AT2R inhibitor, PD123319 under both normoxic and hyperoxic conditions in cells. Finally, the results demonstrated that only the ERK signaling pathway was activated by hyperoxic exposure, but p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) was not affected compared to room-air exposed rats. These data suggest hyperoxia induced lung collagen production via activation of the RAS in MRC-5 cells and in newborn rats, and ERK phosphorylation may be in involved in RAS activation. |
| 關聯: | 86頁 |
| 描述: | 頁數
中文摘要..…………………………………………………………………………..1~2
(Abstract in Chinese)
英文摘要..…………………………………………………………………………..3~5
(Abstract in English)
緒論………………………………………………………………………………...6-23
(Introduction)
研究材料與方法…………………………………………………………………24~33
(Materials and Methods)
實驗結果…………………………………………………………………………34~45
(Results)
討論………………………………………………………………………………46~53
(Discussion)
結論與展望………………………………………………………………………54~55
(Conclusion and Perspective)
參考文獻…………………………………………………………………………56~66
(References)
圖表………………………………………………………………………………67~86
(Tables and Figures)
附錄
論文發表(Publication)
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