星形胶质细胞Cx43缝隙连接通讯在缺血性脑白质损伤中的作用及机制研究

Ischemic White matter disease (WMI) occurs with high incidence whereas the underlying pathological mechanism remains unclear.The latest progress in neuroscience suggests that the key factor of neural regeneration and restoration after axonal injury mainly depends on the astrocyte mediated microenvironment of neural regeneration. Accumulating studies have showed that astrocytes (AST) play an active role in the pathological process of brain white matter ischemic damage. Astrocytes are extensively coupled through Connexin43 mediated gap junction channels which may regulate AST function, the interaction between AST and oligodendrocytes and myelin formation. In the preliminary study, we found that the expression of Cx43 was up-regulated in white matter after ischemic damage, and the injury of myelin sheath and axon could be reduced by administration of CBX, an inhibitor of gap junction channel. According to these results, we speculate that Cx43 and its relevant signal pathways may play active roles in brain white matter injury and myelin repair process after ischemia. In this project, we will study the expression of astrocytic Cx43 gap junctional coupling and its roles in the axonal and myelin injury and repair under white matter ischemic conditions using Cre/loxp gene knockout, immunofluorescence, patch clamp and molecular biology methods in vitro and vivo. The involvement of Cx43 signaling in the damage and repair of neurovascular unit in white matter under ischemic conditions and the underlying mechanism will also be investigated. This project will provide experimental evidence for further understanding the pathogenesis of cerebral white matter ischemic damage and explore potential novel targets for ischemic white matter damage treatment.

缺血性脑白质病变发病率高，病理机制不明。神经科学最新进展提示轴突损伤后的神经再生和修复要依赖于星形胶质细胞起决定作用的神经再生微环境的改善。研究表明星形胶质细胞（AST）活跃地参与了脑白质缺血损伤的病理过程。星形胶质细胞特异性缝隙连接蛋白Cx43及其介导的缝隙连接通讯调节AST功能及AST与少突胶质细胞相互作用，参与髓鞘形成。我们的前期研究亦显示白质缺血后Cx43表达上调，缝隙连接抑制剂能减少髓鞘及轴突损伤，提示Cx43缝隙连接通道及其介导的相关信号通路在脑白质损伤与修复过程中发挥重要作用。本项目采用膜片钳、免疫荧光、Cre/loxp细胞特异性基因敲除等方法，利用离体及白质缺血动物模型，从分子、细胞、整体水平研究星形胶质细胞Cx43在脑白质缺血后的表达、功能及在轴突、髓鞘损伤及再生过程中的作用及其可能的分子信号机制。为进一步理解脑白质缺血的病理机制，探索新的治疗靶点提供实验依据。

研究表明星形胶质细胞（AST）活跃地参与了脑白质缺血损伤的病理过程。星形胶质细胞特异性缝隙连接蛋白Cx43及其介导的缝隙连接通讯调节AST功能及AST与少突胶质细胞相互作用，参与髓鞘形成，但机制不明。本项目利用小鼠双侧颈总动脉狭窄（BCAS）构建低灌注白质损伤模型、离体星形胶质细胞与少突胶质细胞共培养及缺氧缺糖模型，研究星形胶质细胞Cx43在脑白质缺血后的表达、功能及在轴突、髓鞘损伤及再生过程中的作用及其可能的分子信号机制。结果发现低灌注后星形胶质细胞Connexin43表达量明显上调，缝隙连接通讯抑制剂 CBX及MFA干预组及星形胶质细胞Connexin43敲除可保护髓鞘形态及完整性及郎飞氏结结构的完整性，进而促进低灌注后认知功能恢复。研究了星形胶质细胞Cx43调节缺血性脑白质损伤后轴突、髓鞘损伤修复的信号分子机制。结果发现星形胶质细胞Cx43缝隙连接通道抑制降低了缺血后白质区域Glu水平，介导了保护作用。离体缺氧缺糖模型发现Connexin43阻滞剂MFA及CBX可降低慢性缺氧时星形胶质细胞活化及Connexin43的表达、改善OPC分化成熟障碍、减少谷氨酸分泌。这些结果提示Cx43抑制剂对于脑白质缺血性损伤具有保护作用，为进一步理解脑白质缺血的病理机制，探索新的治疗靶点提供实验依据。

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