阻塞性腎病變，是慢性腎臟疾病的主因之一，其特徵是炎症反應的發生、腎小管萎縮及間質性纖維化。纖維化過程中肌成纖維細胞是胞外基質形成主要來源。許多研究指出，肌成纖維細胞主要來自內源性的成纖母細胞或類纖維母細胞、上皮-間質細胞轉換，以及來自骨隨的成纖母細胞。轉化生長因子β1是誘導肌成纖維細胞活化的主要化學因子，但越來越多研究指出，物理特性，譬如基質軟硬度也可能影響其肌成纖維細胞的活化。我們利用膠原蛋白覆蓋聚丙烯醯胺膠體培養系統調節培養基質軟硬度，發現基質軟硬度在轉化生長因子β1誘導的上皮-間質細胞轉換以及成纖母細胞活化扮演很重要的角色。一般認為，細胞外基質的製造與累積可改變基質的軟硬度，進而影響細胞的行為反應。然而，在利用小鼠單側輸尿管阻塞誘發腎臟病變的實驗中，纖維母細胞的活化與胞外基質大量堆積與否似乎沒有明顯的相關性。我們假設這些纖維母細胞的活化是因轉化生長因子β1先誘導成纖維母細胞製造賴氨醯氧化酶 (lysyl oxidase)，藉由賴氨醯氧化酶對膠原蛋白的交聯作用，使得微環境的硬度產生變化。藉此變化，將可影響轉化生長因子β1誘導的上皮-間質細胞轉換或纖維母細胞的活化效率。在本論文中，利用大鼠的腎臟纖維母細胞(NRK49F cells)並給予轉化生長因子β1刺激，我們發現纖維母細胞的活化會受轉化生長因子β1劑量及處理時間而有所不同。培養在軟的膠原蛋白凝膠中，轉化生長因子β1可促使甲型平滑肌纖維與賴氨醯氧化酶的mRNA表現與膠原蛋白凝膠的收縮現象；利用賴氨醯氧化酶抑制劑BAPN可抑制轉化生長因子β1誘導所造成的α-SMA蛋白及mRNA的表現，但不影響賴氨醯氧化酶的mRNA表現。BAPN也抑制了轉化生長因子β1所誘導肌成纖維細胞對膠原蛋白凝膠的收縮能力。利用小鼠單側輸尿管阻塞造成腎病變的實驗發現，BAPN治療可明顯降低慢性腎臟疾病引起之腎小管擴張、胞外基質堆積。BAPN也可降低UUO後期甲型平滑肌纖維的表現，但不影響賴氨醯氧化酶mRNA的變化。綜合以上結果，這項研究將能協助我們了解賴氨醯氧化酶在調控細胞外基質微環境所扮演的角色，並進一步去推論它的細胞生理功能及在腎臟纖維化所扮演的角色。

Obstructive nephropathy, one of the leading causes of chronic kidney disease, is characterized by inflammation, tubular atrophy and interstitial fibrosis. Myofibroblast is the primary source of ECM in the course of fibrosis. Accumulated studies suggested that myofibroblast might come from three sources: endogenous fibroblasts or fibroblast-like cells, epithelial to mesenchymal transition (EMT) and recruitment of fibrocytes from the bone marrow. Although TGF-β1 is suggested as a key inducer for myofibroblast activation, physical effect such as substratum stiffness might also contribute to its activation. Using collagen coated-polyacrylamide gel culture system, our data indicated that TGF-β1 induced EMT and fibroblast activation were substratum stiffness dependent. Both the composition and quantity of extracellular matrix contributes to physical property of the substratum. However, in vivo study with mouse UUO (unilateral ureteral obstruction) model showed that interstitial fibroblast activation preceded the existence of EMT without apparent ECM deposition. We hypothesized that TGF-β1 induced fibroblast activation might be through lysyl oxidase (LOX) eliciting change in microenvironment rigidity. In this study, we showed that NRK49F cells were induced toward myofibroblast activation in response to TGF-β1 within 24 hrs regardless of culturing on stiff culture dish or in/on soft collagen gel. TGF-β1 increased the expression of LOX mRNA whatever cells were cultured. However, BAPN (lysyl oxidase inhibitor) abrogated TGF-β1-induced myofibroblast activation only when cells were cultured in/on soft collagen gel. TGF-β1 increased-fibroblast collagen gel contraction ability was also abolished by BAPN. We then evaluated whether BAPN can diminish the deleterious progression of UUO, a typical model for the tubulointerstitial fibrosis which was an endpoint outcome of chronic renal diseases. BAPN administration reduced the urine accumulation and renal tubular dilation. BAPN alleviated ECM deposition only in the late stage of UUO as assessed by Sirius red staining, masson’s trichrome staining and second harmonic generation by multiphoton confocal microscopy. Finally, BAPN treatment also reduced the α-SMA expression in the late stage of UUO kidney as shown by protein and mRNA level.
Taken this data together, the study facilitates our understanding for the role of lysyl oxidase in modulating microenvironment change and in the pathological development of renal fibrosis.

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