Notch信号参与调控慢性肾病AVF术后血管内膜增生机制的研究

The preferred vascular access for patients with chronic kidney disease and ESRD is an arteriovenous fistula (AVF). AVF failure develops mainly from vascular stenosis created by neointima formation and/or thrombosis. The aim to explore the mechanism and block the progress of neointemal formation is always an important field in Nephrology. The endothelium has a central role in the cascade of neointemal formation events.Activated or injured ECs release inflammatory mediators that trigger platelet aggregation and recruitment of leukocytes to AVF area， it also promote smooth muscle cells migration from the media to the intima as well as proliferation . The overall result is the rapid formation of a neointimal layer over the site of injury. Except that, we found there exist endothelial mesenchymal transition (EnMT) in the process of neointemal formation. EnMT is associated with multiple biological and pathological process such as embryogenesis, organ development, wound healing, tissue regeneration, organ fibrosis and tumor metastasis. However, the mechanism of EnMT involved-neointima formation of AVF remains unknown. This project are focus on the follows : 1,Whether Notch signaling pathway is activated in endothelial cells (ECs) in neointemal formation process?2, Whether ECs acquire the phenotype of fibroblast and the process of EnMT are regulated by Notch signaling pathway? 3,Whether fibroblast specific protein 1(FSP-1), one of the fibroblast marker, is the target gene of Notch signaling pathway? We also want to know whether the process of ECs migrating，losing its barrier function and secreting ECM is regulated by FSP-1 through activation of Notch signaling pathway? 4, To block the Notch signaling pathway, the degree of neointemal formation should be remarkably reduced. The identification of the signaling pathways that lead to activation of EnMT programs is providing theoretical foundation to development of new therapeutic strategies to block neointemal formation, it is also providing experiment evidences to further augment the patency rate of AVF in CKD patient .

慢性肾病发展到终末期肾病主要利用动静脉篓手术（AVF）建立血液通路维持生命，但AVF往往因内膜增生等引起静脉狭窄导致失败。探索新内膜增生机制和干预其进程一直是临床研究的重要领域。内皮细胞在新内膜增生过程中起着关键作用，我们前期研究发现在新内膜增生过程中还存在内皮间质化现象，但内皮间质化在新内膜增生过程中的调节机制目前并不清楚。本项聚焦于：1.新内膜增生过程中内皮细胞Notch信号是否被激活；2. Notch信号是否通过促使内皮细胞获得成纤维细胞表型，发生内皮间质化；3. Notch信号是否通过调节成纤维细胞标记物FSP-1的表达，促使内皮细胞迁移、失去屏障功能，促进新内膜增生；4.靶向阻止内皮细胞Notch信号激活，是否可阻止新生内膜增生过程。认识AVF术后内皮间质化的调节机制，为阐明血管内膜增生的研究奠定理论基础,也为临床上在肾衰竭后AVF术及其他血管成形术后增加通畅率打下应用基础。

慢性肾病发展到终末期肾病主要利用动静脉篓手术（AVF）建立血液通路维持生命，但AVF往往因内膜增生等引起静脉狭窄导致失败。血管新生内膜增生不仅是血管损伤后发生的过度修复过程,也是动静脉瘘术、支架植入术等血管手术以及动脉粥样硬化、高血压等心血管疾病的生理特征。探索新内膜增生机制和干预其进程一直是临床研究的重要领域。内皮细胞损伤、内皮细胞功能障碍、内瘘吻合口处局部血流动力学改变都会引起血管内膜增生。我们前期研究发现在新内膜增生过程中还存在内皮间质化（EndMT）现象，但内皮间质化在新生内膜增生过程中的调节机制目前并不清楚。. 我们通过收集临床病人AVF标本，建立小鼠CKD模型和AVF模型，研究了内皮细胞内皮间质化情况，并进一步在细胞水平研究Notch信号激活与EndMT的相关性。我们发现慢性肾病增加了AVF部位TGF-beta1、整合素beta3、成纤维细胞标志分子FSP-1的表达，FSP-1启动子区域存在Notch信号下游转录因子RBP-JK的结合位点。TGF-beta可以促进内皮细胞Notch信号激活，促进了FSP-1的表达，造成内皮细胞间质化，内皮细胞细胞连接受到破坏，引起血管新生内膜增生。此外，整合素beta3通过对Notch信号的激活引起内皮间质化，从而促进血管AVF术后新生内膜增生。Rapamycin则可通过对Notch信号的阻断阻止内皮间质化进程。通过整合素beta3的敲低或Rapamycin处理，可阻断内皮细胞Notch信号通路，阻止内皮间质化进程,从而对抑制临床上AVF术后血管内膜增生，也对改善慢性肾病患者AVF术后血管通畅率提供了实验依据。