- 學位論文
歐室塵蟎過敏原 Der P 2 誘導人類肺臟纖維 細胞增殖與膠原蛋白過度表現之研究
Studying House dust mite allergen Der P 2 induced proliferation and collagen overexpression of human lung fibroblast MRC-5.
摘要
肺纖維化疾病的發病機制目前傾向於肺損傷所導致引起，其次是發炎反應，進而造成纖維化的產生，接著會導致纖維化的增生。 這說明了慢性損傷和發炎跟纖維化之間的關係，在肺纖維化中扮演了重要作用。 歐洲室塵蟎第二群過敏原(Dermatophagoides pteronyssinus group 2 allergen, Der p 2)為不具有蛋白水解酶特性的主要塵蟎過敏原之一，這類過敏原對於纖維化細胞的影響知識有限。不過有研究顯示利用重組蛋白過敏原Der p 2(DP2) 透過激活磷酸肌醇3激酶(PI3K)和促分裂原活化蛋白激酶（MAPK）途徑，也被稱為調解毒蕈鹼受體誘導的途徑，導致肺纖維細胞增殖。然而，DP2誘導的呼吸道纖維細胞增殖之機制仍未完全清楚。本研究利用免疫轉漬 (western blot)、聚合酶鏈反應(PCR)跟即時聚合酶鏈鎖反應(Real-time RT-PCR)等分析方法，發現以DP2刺激人類肺纖維母細胞MRC-5，會導致其α-平滑肌肌動蛋白(αα-SMA)、纖維連接蛋白 (Fibronectin)、第一型膠原蛋白 (Collagen I)的蛋白跟mRNA有顯著的增加。接著進一步利用激酶活化分析相關的訊息傳導的途徑，發現PI3K/AKT、ERK/MAPK (ERK)、c-Jun氨基末端激酶 (JNK) 和p38 MAPK (p38) 等蛋白激酶的磷酸化都有增強的現象。再藉由專一性抑制劑的實驗分析，發現DP2主要是透過活化細胞核轉錄因子β-catenin以及活化激活促分裂原活化蛋白激酶，導致α-平滑肌肌動蛋白、纖維連接蛋白以及第一型膠原蛋白的表現。我們的研究結果也顯示DP2會誘導人類肺纖維細胞MRC -5的增殖現象。
並列摘要
Der p 2 (DP2), a Dermatophagoides pteronyssinus group 2 allergen, is well-known to cause airway hypersensitivity and highly associates with induction and progression of asthma. Previous studies have shown that DP2 activates several mitogen-activated protein kinase (MAPK) pathways, which is also known to mediate muscarinic receptor-induced human lung fibroblast proliferation. Airway fibroblast proliferation and the consequent fibrosis is an important pathological characteristic of chronic inflammatory and obstructive airway diseases such as asthma, COPD and Idiopathic pulmonary fibrosis - IPF. However, whether DP2 induces proliferation of airway fibroblast and the underlying mechanisms remain sketchy. The present study demonstrated that DP2 increased expression of type I-collagen, fibronectin and alpha-SMA in both mRNA and protein level. In parallel, kinase activation assay revealed that that DP2 enhanced phosphorylation of PI3K/AKT, MAPKs including mitogen-activated protein kinase kinase kinase 1 (MEKK1), c-Jun N-terminal kinase 1 (JNK1) and p38 MAPK (p38).The levels of nuclear β-catenin were increased after treatment by DP2.β-catenin also contributed to DP2-induced type I-collagen, fibronectin and alpha-smooth muscle actin (alpha-SMA). Further, DP2 treatment induced significant proliferation of human lung fibroblast MRC-5 by using MTT assay.Taken together, our findings indicate that DP2 induces proliferation and collagen overexpression of airway fibroblast MRC-5 which may attribute to activation of MAPK and PI3K/AKT pathway and provide a possible mechanism for allergen-induced airway fibrosis.