臺灣博碩士論文加值系統

關於特發性肺纖維化 (IPF) 的作用機轉至今仍然尚未明朗。受 IPF 折磨的患
者除了鮮少有藥物可完善的治療外，他們的肺功能也會隨著 IPF 病情進展而惡
化，最終引發呼吸衰竭而需要仰賴呼吸器來延遲不可避免的死亡。肺損傷後失衡
的上皮細胞間質轉化 (EMT) 作用可能為造成 IPF 的導因之一，其最終結果為肌
纖維母細胞聚積，並造成過多細胞外基質 (ECM) 沉積。乙型轉化生長因子
(TGF-β) 是主要的前纖維化細胞激素之一，先前研究顯示 TGF-β 能誘導肺上皮
細胞的 EMT 作用而輔助肌纖維母細胞的累積。此外，有證據顯示 Hippo 訊息傳
遞路徑中的轉錄因子，Yes-相關蛋白 1 (YAP1) 活性在癌症相關纖維母細胞中是
提升的。而當中的 YAP1 透過 ROCK 反應路徑來硬化 ECM。綜合以上研究，雖
然對於 IPF 反應路徑有初步見解，但 ROCK-YAP1 反應路線是否參與在 TGF-β
誘導的 EMT 作用在肺上皮細胞還未有明確見解。因此本論文的假說為肺上皮細
胞 ROCK-YAP1 訊息傳遞路徑參與在 TGF-β 誘導的 EMT 中。起初，我們發現博
來黴素 (Bleomycin) 經氣管刺激的老鼠肺纖維化組織當中，YAP1 磷酸化的表現
是下降的。接著，為了在細胞實驗模擬 IPF，我們以 TGF-β 刺激 A549 肺上皮細
胞產生 EMT 反應 24 小時，並在結果發現間質標的纖連蛋白 (fibronectin) 與第
一型膠原蛋白 (type I collagen) 的表現量是提升的。進一步的，在 A549 經 TGFβ 誘導的 EMT 作用中，ROCK1 與 YAP1 也被證實能貢獻於間質標的生成。當瞭
解以上訊息傳導物質都與 EMT 有關聯後，下一步就是釐清他們彼此的傳導路徑。實驗結果發現 TGF-β 受體抑制劑可抑制 TGF-β 刺激肺泡上皮細胞 ROCK1 的磷
酸化、YAP1 的去磷酸化以及 YAP1 luciferase 活化。此外，ROCK 抑制劑也可減
少 TGF-β 誘導的 YAP1 去磷酸化以及 YAP1-luciferase 活化，代表著 YAP1 是在
ROCK1 活化後的下游傳遞物質。綜合以上研究，由於 IPF 是不可逆的疾病，因
此每一條發現造成 IPF 的路徑都是可貴的。本論文首次證實了 TGF-β-ROCK1-
YAP1 傳遞路徑在肺泡上皮細胞的 EMT 反應中，且此路徑有可能貢獻於 IPF 的
病理進程。

The mechanisms leading to idiopathic pulmonary fibrosis (IPF) are still unclear.
Patients suffering from IPF not only have limited pharmacological drugs to effectively
ease the burden of disease, their pulmonary function also deteriorates with progressive
lung scarring due to the nature of IPF pathogenesis, which at the end may result in
respiratory failures requiring mechanical ventilation to prolong the inevitable death.
Dysregulated epithelial-mesenchymal transition (EMT) after lung injury has been
suggested to be responsible for IPF formation, with accumulation of myofibroblast
producing excessive extracellular matrix (ECM) as the end product. Transforming
growth factor-beta (TGF-β), a major pro-fibrotic cytokine, was hinted to promote
myofibroblast deposition through the EMT process. Interestingly, yes-associated
protein 1 (YAP1), a member of Hippo pathway, was upregulated in cancer-related
fibroblasts that stiffened the ECM dependent on the rho-associated coiled-coil
containing protein kinase (ROCK) pathway. However, the signal transduction of TGFβ-ROCK-YAP1 in lung alveolar epithelial cells during TGF-β-induced EMT remains
unknown. Our hypothesis is ROCK-YAP1 pathway participated in the TGF-induced
EMT in lung epithelial cells. In this study, we observed YAP1 activation in fibrotic lung
tissues of bleomycin-treated mice. Next, we created an IPF model in A549 type II lung
epithelial cell by inducing EMT by TGF-β for 24 hours, resulting in upregulation of mesenchymal markers, fibronectin and type I collagen. Next, we demonstrated TGF-β,
ROCK1, and YAP1 all contributed to the formation of mesenchymal markers in TGFβ-induced EMT in A549 cells. Subsequently, we looked into the signal transduction
between TGF-β, ROCK1, and YAP1. Upon TGF-β exposure, ROCK1 phosphorylation,
YAP1 activation, and YAP1-luciferase activity were suppressed by TGF-β type I
receptor blocker. On the other hand, YAP1 dephosphorylation and YAP1 luciferase
activity were inhibited by pretreating A549 with ROCK inhibitor before TGF-β
stimulation. In conclusion, IPF is an irreversible interstitial disease, which underlines
the importance of every newly found pathway leading to IPF. In the current study, we
were the first to describe ROCK1-YAP1 pathway during TGF-β induced EMT in lung
alveolar epithelial cells, which may play a role driving fibrogenic formation of IPF.

Abbreviations ............................................................................................................... iii
Acknowledgment ........................................................................................................... v
I:中文摘要 ................................................................................................................. viii
II: English Abstract ........................................................................................................ x
III. Introduction .............................................................................................................. 1
III-1. Idiopathic pulmonary fibrosis (IPF) ........................................................................ 1
III-2 Epidemiology of IPF ................................................................................................... 1
III-3 Pathogenesis leading to IPF ....................................................................................... 2
III-4 Epithelial-mesenchymal transition (EMT) ............................................................... 2
III-5 Transforming growth factor-beta (TGF-β) .............................................................. 3
III-6 Rho-associated protein kinase (ROCK) signaling pathway.................................... 5
III-7 Yes-associated protein 1 (YAP1) ............................................................................... 6
IV. Hypothesis and Objectives ....................................................................................... 8
V. Materials & Methods ................................................................................................. 9
V-1 Study design .................................................................................................................. 9
V-2 Generation of bleomycin-induced IPF animal model ............................................... 9
V-3 Masson Trichrome Staining ........................................................................................ 9
V-4 Immunohistochemistry (IHC) ................................................................................... 10
V-5 Cell culture .................................................................................................................. 11
V-6 SDS-PAGE and Western blot .................................................................................... 12
V-7 RNA Interference and Transfection ......................................................................... 14
V-8 Luciferase Reporter Assay ........................................................................................ 15
V-9 Statistical Analysis...................................................................................................... 16
V-10 List of material, equipment, and software utilized ................................................ 16
VI. Results.................................................................................................................... 21
VI-1 Increased YAP1 activation in lung tissues of bleomycin-treated mice ................ 21
VI-2 Induction of EMT in A549 lung epithelial cells by TGF-β stimulation ............... 21
VI-3 Effect of TGF-β receptor inhibition on TGF-β-induced EMT in A549 lung
epithelial cells ..................................................................................................................... 22
VI-4 Effect of ROCK inhibition on TGF-β-induced EMT in A549 lung epithelial cells
............................................................................................................................................. 22
VI-5 Effect of silencing YAP1 gene expression on TGF-β-induced EMT in A549 lung
epithelial cells ..................................................................................................................... 23
VI-6 ROCK1 as a downstream effector during TGF-β-induced EMT in A549 lung
epithelial cells ..................................................................................................................... 23
VI-7 YAP1 acts as the downstream effector of ROCK1 during TGF-β-induced EMT
in A549 lung epithelial cells .............................................................................................. 24
VI-8 Luciferase reporter assay proved YAP1 as the downstream effector of ROCK1
activation during TGF-β-induced EMT in A549 lung epithelial cells .......................... 24
VII. Discussion ............................................................................................................ 26
VIII. Conclusion .......................................................................................................... 34
IX. Figures ................................................................................................................... 35
X. References ............................................................................................................... 50

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