巴拉刈誘發人類肺纖維母細胞株 MRC-5 產生膠原蛋白之分子機制

The Molecular Mechanism of Paraquat-induced Collagen Production in Human Lung Fibroblast MRC-5 Cells

藍耀東(Yaw-Dong Lang)

指導教授 ： 汪棱芳

臺北醫學大學/醫學院/醫學科學研究所/碩士(2006年)

摘要

巴拉刈（Paraquat, 1,1'-dimethyl-4,4'-bipyridium; methyl viologen）為仿間常用之除草劑，根據台灣地區藥物諮詢檢驗中心的統計，巴拉刈中毒的致死率為所有中毒物質中最高者(Chen et al., 2005)。因此，本論文以人類正常肺纖維母細胞MRC-5為細胞模式，排除巴拉刈之腎毒性造成腎素-血管張力素系統之影響，來探討巴拉刈導致肺臟纖維化之機制。 由結果顯示，巴拉刈刺激MRC-5細胞誘導膠原蛋白表現是受到血管張力素II的調控，藉由real-time RT-PCR發現細胞內血管張力素轉化?第一型、第二型基因表現均下降，但糜蛋白?(chymase)的基因表現卻上升，因而推測chymase可能是扮演催化血管張力素II生成的角色，之前的報導均認為chymase是由mast cell所分泌，這是首次發現纖維母細胞亦能分泌chymase並調控血管張力素II之產生，而血管張力素II的表現會被chymase抑制劑(chymostatin)所抑制。 另ㄧ方面，細胞在給予巴拉刈刺激後會進一步誘發其血管張力素II第一型受體蛋白(angiotensin II typeⅠreceptor, AT1R)表現及活化其下游的甲型轉型細胞生長因子與結締組織生長因子之基因與蛋白表現；我們亦發現非選擇性血管張力素II受體抑制劑(saralasin)確實會抑制細胞內甲型轉型細胞生長因子、結締組織生長因子與膠原蛋白的表現。此外，實驗結果發現巴拉刈所誘導膠原蛋白增加僅促進細胞內cathepsin L的基因表現增加及酵素活性，加入cathepsin L抑制劑(Z-Phe-Phe-CH2F)證實cathepsin L確實扮演水解膠原蛋白的角色，以維持其細胞內膠原蛋白之恆定性。 綜合以上實驗結果，本論文發現巴拉刈會藉由chymase來催化人類正常肺臟纖維母細胞(MRC-5)內血管張力素II的產生，同時藉活化其下游路徑之TGF-β1、CTGF表現並誘導膠原蛋白的生合成。

關鍵字

巴拉刈 ； 膠原蛋白 ； 纖維化 ； 選擇性 ； 細胞株 ； 恆定性 ； 致死率 ； 除草劑

並列摘要

Paraquat dichloride (1,1-dimethyl-4,4-bipyridilium dichloride; methyl viologen) is an effective and widely used herbicide. According to epidemiological evidence in the National Poison Center in Taiwan, paraquat poisoning has suggested that was the leading cause of poisoning-induced death in Taiwan. In this study, the normal human fetal lung fibroblast cell line (MRC-5) was used to study the mechanism of paraquat-induced pulmonary fibrosis for eliminating the effect of paraquat nephrotoxicity on renin-angiotensin system (RAS). The results showed that paraquat-induced collagen expression in MRC-5 was upregulated by angiotensin (ANG) II. However, the responsible enzyme for the conversion of ANG I to ANG II was chymase and the up-secretions of ANG II and collagen were inhibited by chymostatin. On the other hand, this study also demonstrated the induction of transfoming growth factor (TGF)-β1 and connective tissue growth factor (CTGF) by paraquat was mediated via activation of the angiotensin II type 1 receptor (AT1R) and inactivation of the angiotensin II type 2 receptor (AT2R). The unselective AT1R and AT2R antagonist saralasin also attenuated the increases in TGF-β1, CTGF and collagen synthesis induced by paraquat administration in MRC-5. Furthermore, we also showed that paraquat-induced collagen was hydrolyzed by cathepsin L in MRC-5 cell. We concluded that ANG II induced-collagen overexpression in paraquat-administrated lung fibroblast was mediated via the activity of chymase rather than angiotensin I converting enzyme I.

並列關鍵字

paraquat ； collagen ； fibrosis ； system ； taiwan

參考文獻

Addo E, Poon-King T. Leukocyte suppression in treatment of 72 patients with paraquat poisoning. Lancet. 1986;1:1117-20.

Arrieta O, Guevara P, Escobar E, Garcia-Navarrete R, Pineda B, Sotelo J. Blockage of angiotensin II type I receptor decreases the synthesis of growth factors and induces apoptosis in C6 cultured cells and C6 rat glioma. Br J Cancer. 2005;92:1247-52.

Arteaga CL. Inhibition of TGF-beta signaling in cancer therapy. Curr Opin Genet Dev. 2006;16:30-7.

Belova LA. Angiotensin II-generating enzymes. Biochemistry (Mosc). 2000 ;65:1337-45.

Bhatt N, Baran CP, Allen J, Magro C, Marsh CB. Promising pharmacologic innovations in treating pulmonary fibrosis. Curr Opin Pharmacol. 2006;6:284-92.