腎臟纖維化特徵為在組織間隙中有大量胞外基質堆積而使器官硬化。先前研究指出主要原因為來自於腎小管上皮細胞經轉化生長因子beta 1刺激下進行上皮-間質細胞轉換而促使肌纖維母細胞大量出現。另一方面，細胞所處的基質軟硬度會調控細胞生物功能，如幹細胞分化，癌細胞表現，細胞生死及生長因子的作用。本篇論文主要在探討基質軟硬度是否可調控轉化生長因子beta 1誘導上皮-間質細胞轉換。我們利用聚丙烯醯胺凝膠製造出不同軟硬度的基質，再將豬的腎臟上皮細胞株(LLC-PK1)及從小鼠腎臟取出初級培養的近端腎小管上皮細胞 (mPTs) 培養在其上。在一般培養皿上，給予轉化生長因子beta 1可降低上皮細胞E-cadherin的量，增加alpha-SMA，fibronectin及beta 1 integrin的表現。但當細胞培養在軟性基質上，不管是對細胞型態，骨架分佈或相關蛋白表現上的作用皆有被明顯抑制。更重要的是，當mPTs 種在軟性基質(<2415 Pa)七天後，仍能保有原先在體內管狀立體的結構及功能，但硬性基質則會促使其大量增生。在實驗室先前的研究中，利用了中和性 beta 1 integrin抗體的抑制作用指出beta 1 integrin在轉化生長因子beta 1所誘導的上皮-間質細胞轉換過程扮演重要的角色。在本論文中，我們發現細胞在軟性基質下，beta 1 integrin的蛋白質及訊息核糖核酸量遠比在硬性基質還低，且是透過轉錄及後轉錄機制所調控。最後，我們也發現不須轉化生長因子的存在，軟性基質即可抑制Smad3、ERK磷酸化，甚至是ERK的表現。綜合以上結果，我們推論軟性基質會抑制細胞 beta 1 integrin的表現量，進而抑制整個上皮-間質細胞轉換的過程。這研究將能協助我們了解外在組織軟硬度如何調控細胞生理功能及在腎臟纖維化中所扮演的角色。

Recent studies indicate that substratum stiffness regulates cell behavior, i.e. stem cell fate, cancer cell phenotype, apoptosis and growth factor-induced cell differentiation. However, whether substratum stiffness modulates epithelial-mesenchymal transition (EMT) induced by transforming growth factor beta 1 (TGF-beta 1) has not been studied. To elucidate the role of substratum stiffness in TGF-beta 1-induced EMT, we employed type I collagen-coated polyacrylamide gel of various stiffness. We observed that TGF-beta 1-induced EMT in LLC-PK1 cells and primary culture of mice proximal tubular cells (mPT) were manifested by upregulation of alpha-SMA, fibronectin, beta 1 integrin and downregulation of E-cadherin. TGF-beta 1-induced EMT changes could be abrogated by low substratum stiffness in both cell line and strain. Interestingly, unlike the phenotype of primary mPT cells cultured on stiffer substratum, primary mPT cells cultured under stiffness of lower than 2415 Pa did not spread out but maintained tubular structure throughout 7 days regardless of the presence of TGF-beta 1. To further elucidate the mechanism, we found that TGF-beta 1-induced EMT could be blocked by neutralizing beta 1 integrin antibody. On the other hand, low substratum stiffness downregulated beta 1 integrin protein and mRNA level regardless of the presence of TGF-beta 1 via transcriptional and post-transcriptional regulation. Finally, low substratum stiffness reduced phosphorylation of Smad3, ERK and expression of ERK regardless of the presence of TGF-beta 1. Taken these data together, low substratum stiffness inhibits the expression of beta 1 integrin, which might mediate TGF-beta 1-induced EMT. On the other hand, substratum stiffness alone modulates different signal pathways which were related to TGF-beta 1 signal. This study facilitates our understanding of how substratum stiffness regulates the normal physiological function and the pathological development of renal fibrosis.

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