以Smurf2为靶点调控移植肾间质纤维化形成的机制及干预研究

Renal allograft interstitial fibrosis is the key factor affecting the long-term survival after renal allograft transplantation, but the mechanism is still unknown. Our studies demonstrated that E3 ligase- - Smurf2 played an important role in renal interstitial fibrosis induced by obstructed nephropathy through regulating activity of transforming growth factor-beta. But for its function and mechanism in renal allograft interstitial fibrosis, have never been reported at home and abroad. We found recently that the expression of Smurf2 was significantly up-regulated in renal tubular epithalial cells of recipients with renal allograft interstitial fibrosis. Accordingly, we put forward the hypothesis: Smurf2 maybe play a critical role in the formation of renal allograft interstitial fibrosis through regulating phenotype of renal tubular epithelial cells. To test this hypothesis, this project will explore effects and mechanisms of Smurf2 in the formation of renal allograft interstitial fibrosis mediated by tumor necrosis factor-alpha through a rat renal allograft transplantation and the normal rat renal tubular epithelial cells model, using PCR, Western blot and drug interference and other means. On this basis, we will in-depth study the protective effects and mechanisms of a new type of proteasome inhibitor- - Bortezomib in the formation of renal allograft interstitial fibrosis. The expected results of this study will provide new clues and targets for prevention and treatment of renal allograft interstitial fibrosis from the regulation of protein degradation point of view.

移植肾间质纤维化是影响肾移植术后长期存活的关键因素，其机制至今不明。我们研究证实E3泛素连接酶- - Smurf2可通过调节转化生长因子-beta信号活性而在梗阻性肾病致肾间质纤维化形成中起重要作用；但对其在移植肾间质纤维化中的作用及机制，迄今未见报道。我们新近发现Smurf2在移植肾间质纤维化患者肾小管上皮细胞中表达显著升高，因此提出：Smurf2可能通过调控肾小管上皮细胞表型进而在移植肾间质纤维化形成中起关键作用。为验证该假说，本课题拟通过同种异体肾移植大鼠和正常大鼠肾小管上皮细胞模型，采用PCR、免疫蛋白印迹及基因干扰等手段，以肿瘤坏死因子-alpha为介质，探讨Smurf2在移植肾间质纤维化形成中的作用及机制。并在此基础上，深入探讨新型蛋白酶体抑制剂- - Bortezomib在其中的保护作用及机制。本课题预期成果为从调控蛋白质降解角度研发防治移植肾间质纤维化形成的措施提供新线索和靶标。

移植肾间质纤维化是引起慢性移植肾失功进而影响移植肾预后的关键因素，而肌成纤维细胞聚集作为移植肾间质纤维化的决定性效应细胞，其来源及激活机制至今未明且已成为当今学者瞻目的研究热点。本项目从上皮细胞-间质细胞转分化（Epithelial-mesenchymal transition, EMT）现象入手，运用基因转染等技术，并通过蛋白免疫印迹、实时定量RT-PCR等方法，从慢性移植肾失功患者、同种异体肾移植大鼠模型及人肾小管上皮细胞三个水平，深入探讨肿瘤坏死因子-α（Tumor necrosis factor α, TNF-α）、smad泛素化调节因子2（Smurf2）在移植肾间质纤维化形成中的作用及相关机制，并在此基础上进一步探讨硼替佐米（Bortezomib）在其中的影响及可能的机制。本研究首次在人与大鼠动物模型中证实Smurf2的表达变化存在于移植肾间质纤维化形成过程中，并与移植肾间质纤维化形成密切相关；并证实TNF-α可通过诱导Akt信号传导通路活化上调Smurf2的表达，进而促进肾小管EMT和移植肾间质纤维化的形成。此外，本研究还首次在同种异体肾移植大鼠模型中证实了Bortezomib可通过抑制Smurf2表达及移植肾肾小管EMT现象进而延缓甚至逆转移植肾间质纤维化形成，并指出Bortezomib的该作用与其抑制细胞内Akt等信号传导通路的活性相关。本项目的研究结果不仅有助于从分子水平深入认识移植肾间质纤维化的形成机制，同时首次提出对Bortezomib临床应用扩展的探索、并揭示其在拮抗移植肾间质纤维化形成的作用及可能的机制，而且首次提出可以从调控Smurf2表达的角度设计并开发防治移植肾间质纤维化进展的新措施。