Notch-TGF-beta/Smad信号调节脊髓损伤后星形胶质化和瘢痕形成的作用机制研究

The spinal cord injury is a severe debilitating trauma problem in the hospital currently. The remarkable gliosis and glial scar formation in injured area obstruct axonal regeneration obviously, and it remains a key question how to deal with inhibition of glial scar on axonal regeneration during the spinal cord injury and recovery. Evidence has shown that glial scar is mainly generated from activated astrocytes and microglial cells, and Notch signaling pathway may critically involve in glial functional activation. Our study has shown that myeloid-specific blockade of Notch signaling pathway by RBP-J knockout (RBP-j-KO) attenuates microglial M1 polarization and inflammation, promote axonal regeneration and improve motor function after spinal cord injury. Our preliminary experiment results further indicated that application of specific Notch signaling pathway inhibitor in the cultured astrocytes resulted in decease of TGF-beta, Smad2 and CS56 expression levels. We thus hypothesize that Notch-TGF-beta/Smad signaling may function as a key regulator in the astrogliosis and glial scar formation. In this project, astrocyte-specific blockage of Notch signaling by RBP-j-KO, the spinal cord injury animal model, cell culture of astrocytes and specific inhibitors will be applied to study roles of Notch signaling pathway in regulation of astrocytic activation or astrogliosis, glial scar formation, axonal regeneration, and possible TGF-beta/Smad signal mediating mechanism. It is hopefully expected to find or to provide a new manipulation target for the spinal cord injury.

脊髓损伤是目前临床面临的难题，损伤区显著的胶质细胞活化和瘢痕形成是阻碍轴突再生的重要原因，如何解除胶质瘢痕对轴突再生的抑制是脊髓损伤修复的关键问题。资料表明活化星形胶质和小胶质细胞是胶质瘢痕的主要来源，而Notch信号是胶质细胞功能的重要调节因子。我们前期研究发现巨噬细胞特异性Notch信号阻断能降低脊髓损伤后小胶质细胞M1极化和炎症反应、促进轴突生长和运动恢复，预实验结果发现活化星形胶质细胞给予Notch阻断剂可抑制TGF-beta、Smad2和CS56表达。我们推测Notch-TGF-beta/Smad信号可能是星形胶质化反应和瘢痕形成的重要调节途径。本项目拟构建条件性RBP-j敲除小鼠，通过脊髓损伤动物模型、细胞培养与抑制剂干预等方法，研究阐明Notch信号调节星形胶质细胞活化增殖、胶质瘢痕形成、轴突再生以及TGF-beta/Smad信号激活介导机制，为脊髓损伤治疗提供新的干预靶点。

脊髓损伤与修复是临床面临的难题，针对脊髓损伤后胶质细胞活化和瘢痕形成阻碍轴突再生修复的关键问题，本项目提出了Notch-TGFbeta/Smad信号调节星形胶质化反应和瘢痕形成的研究假设。计划内容是通过构建条件性RBP-j-KO小鼠、脊髓损伤模型、细胞培养与抑制剂干预等方法，研究明确Notch信号调节胶质细胞活化和炎症反应、瘢痕形成和轴突生长作用，阐明Notch-TGFbeta/Smad效应途径的作用机制，希望为探索脊髓损伤干预治疗方法提供新的证据。.本课题获得以下研究进展与结果：I）星形胶质细胞特异性Notch敲除小鼠培育成功；II）通过小鼠脊髓损伤（压迫损伤）模型实验和BMS评分比较，表明阻断Notch信号能够改善脊髓损伤后的运动功能恢复；III)脊髓损伤模型反应性星形胶质细胞上调proNGF表达水平；IV）Notch-PI3K-AKT信号激活促进反应性星形胶质细胞上调其炎症因子的合成水平；V）反应性星形胶质细胞能够显著上调表达和向细胞外释放重要的非经典Wnt信号通路配体Wnt5a分子；VI）阻断Notch信号通路下调脊髓损伤后炎症因子和上调抗炎因子的蛋白表达水平。目前在分析Notch信号阻断对于脊髓神经元生存与细胞凋亡、神经突起（轴突）生长、星形胶质细胞和小胶质细胞活化、少突胶质细胞和髓鞘等神经化学指标的影响。.本项目按计划完成主要研究内容，研究结果发表通讯作者论文5篇，其中SCI研究论文1篇，中文期刊论文4篇。修稿中1篇。达成项目预期的研究目标。本课题研究的主要发现是选择性阻断（抑制）星形胶质细胞Notch信号激活能够显著减轻脊髓损伤炎症反应和促进运动功能恢复作用，初步阐明Notch-PI3K-AKT信号通路介导胶质细胞活化和炎症反应的新机制，为探索脊髓损伤治疗方法提供了新的潜在靶点，选择性干预Notch-PI3K-AKT信号通路调节脊髓损伤胶质细胞活化和继发性炎症反应的治疗意义，有待进一步深入研究阐明。