Tim-3介导小胶质细胞表型转换在脑出血后继发性脑损伤中的作用及其机制研究

Inflammation is one of key causes in secondary brain injury after intracerebral hemorrhage (ICH). The activation of microglia （Phenotypic transformation from M0 to M1）is one initial factor of inflammation. In recent years, immunomodulatory effects mediated by Tim-3 draw more and more attention. According to the related reports and the pre-experiment results, we put forward a scientific hypothesis that Tim-3 mediates the activation of microglia and promotes the inflammatory response, thus involving in the secondary brain injury after ICH. To confirm this hypothesis, we perform this study in four aspects: firstly, exploring the relationship between Tim-3 expression and activation of microglia by construction of in vivo and in vitro models of ICH; secondly, verifing the role of Tim-3 mediated activation of microglia in brain injury after ICH through siRNA, overexpression and other treatments and combination a variety of indicators of brain injury; thirdly, exploring the molecular mechanism of Tim-3 mediated activation of microglia in ICH with analyzing proteins of signaling pathway of Tim-3; Lastly, collection of blood and CSF samples of ICH patients and correlation analysis between Tim-3 level and clinical prognosis. This project will provide reference and theoretical basis for depth analysis of pathophysiology in secondary brain injury after ICH; at the same time, can provide a new reference index for the prognosis of patients with ICH.

炎症是脑出血后继发性脑损伤关键原因之一；小胶质细胞激活（M0向M1表型转换）是炎症起始因素之一，其机制仍不完全清楚。近年来，Tim-3介导免疫调节作用受到越来越多关注，根据相关报道及预实验结果，我们提出一个科学假说：Tim-3介导小胶质细胞激活，促进炎症反应，参与脑出血后继发性脑损伤。为证实该假说，我们拟从四个方面开展研究:一是构建脑出血体内体外模型，探究Tim-3表达水平变化及其与小胶质细胞激活的关系；二是通过siRNA，过表达等方法并结合多种脑损伤指标验证Tim-3介导小胶质细胞激活在脑出血后脑损伤中的作用；三是分析Tim-3信号通路相关蛋白，探究Tim-3介导小胶质细胞激活在脑出血后脑损伤中的分子机制；四是收集脑出血病人血液及脑脊液，分析Tim-3表达水平与脑出血病人预后的相关性。本项目将为深入解析脑出血后继发性脑损伤病理生理机制提供理论基础，并为预测脑出血病人预后提供新的参考指标。

目的探讨Tim-3在脑出血(ICH)继发性脑损伤(SBI)小胶质细胞极化中的关键作用。采用自体全血注射大鼠右侧基底神经节建立颅内出血模型。用氧-血红蛋白处理原代培养的小胶质细胞，体外模拟脑出血。在本实验中，利用Tim-3和重组人Tim-3的特异性siRNA在体内和体外进行研究。脑缺血后Tim-3在脑内升高，主要分布在小胶质细胞中，神经元和星形胶质细胞中不明显。然而，siRNA阻断Tim-3可显著减少炎症因子的分泌、神经元变性、神经元细胞死亡和脑水肿。同时，Tim-3的下调促进了ICH后小胶质细胞表型由M1向M2的转化。此外，上调Tim-3可增加ICH后Tim-3与Galectin-9 (Gal-9)的相互作用，激活toll样受体4 (TLR-4)通路。Tim-3表达增加可能与HIF-1α的激活有关。Tim-3可能是通过Tim-3/Gal-9和TLR-4信号通路导致脑出血后SBI的神经炎症和小胶质细胞极化之间的重要联系。