TRPM7在神经细胞缺血损伤中的作用及DREAM对TRPM7调控机制研究

Neuronal ischemia injury is a primary cause of neurological impairment in ischemic stroke patients. Cerebral ischemia injury is mainly associated with the excitotoxic action induced by glutamate excitotoxicity (NMDA receptor mediated) pathway. But pharmacological agents that disrupt the excitotoxic pathway have only led to disappointing clinical trials. TRPM7 is Ca2+-permeable divalent cation channel protein and is associated with neuronal cell death under ischemic stresses. The activation of TRPM7 is a NMDA-independent pathway that significantly contributes to the pathological Ca2+ overload. It is revealed that DREAM is a Ca2+-binding modulator of channel proteins. Our study and other studies have showed the high expressions of DREAM and TRPM7 in the central and surrounding areas of the infarction. Based on the Ca2+-binding characteristics of DREAM and the Ca2+-permeable property of TRPM7, we propose that both DREAM and TRPM7 being involved in ischemic damage together，owing to possible functional relationship between them. Therefore, we will upregulate and downregulate the expression of TRPM to study the mechanism of TRPM7-mediated neuronal damage in vitro (oxyzen-glucose deprivation fowllowed by reoxygenation cell model) and in vivo (TRPM7 knock-out mice model). Then, we will further investigate that mechanism of DREAM-mediated modulation of TRPM7 in the process of cerebral ischemic induced injury. Our study would promote the deep understanding of exact modulating mechanism of TRPM7 expression and provide a new target for further development of neuroprotection drugs on stroke.

神经元缺血损伤是脑卒中引起脑功能障碍的核心之一，NMDA 受体介导的“兴奋性毒性”作用是导致损伤的重要原因，但NMDA受体拮抗剂的临床试验未能显示有效。TRPM7是不依赖NMDA的通道蛋白，激活后引起细胞内Ca2+持续超载，导致恶性氧化应激。目前认为DREAM是多种通道蛋白的调控因子，相关研究和前期实验提示梗死区DREAM和TRPM7蛋白表达均显著增加。针对DREAM作为Ca2+结合蛋白的特性和TRPM7对Ca2+的通透性的特点，我们提出假说：二者可能存在一定的功能相关性，共同参与神经细胞缺血损伤过程。我们采用在体外培养神经元细胞中上调或下调TRPM7表达，同时应用TRPM7基因敲除小鼠，以阐明TRPM7在缺血损伤中的作用，在此基础上探讨DREAM与TRPM7的结合及对TRPM7通道生化功能特性的调控机制。本课题将进一步拓宽对中枢TRPM7通道功能的认识，为脑卒中治疗药物研发提供新靶点。

本研究计划拟阐明TRPM7在神经细胞缺血损伤中的作用，探讨DREAM与TRPM7是否结合及对TRPM7通道生化功能特性的调控机制。首先，我们建立了原代培养的皮层神经元的氧糖剥夺(oxygen-glucose deprivation, OGD)模型和成年大鼠MCAO（Middle cerebral artery occlusion）模型，通过分子生物技术检测到在神经细胞缺氧时TRPM7的蛋白和mRNA均表达增加，推测TRPM7参与神经细胞缺氧损伤；进而探讨TRPM7在大鼠MCAO模型再灌注72小时内的表达变化，认识到在脑缺血再灌注24-48小时之间是TRPM7的表达高峰；其次，分别使用TRPM7的抑制剂2-APB、香芹酚调控TRPM7，结果显示可减少缺血损伤时神经元的凋亡、明显减少梗死体积以及减轻神经功能障碍；现已探测到DREAM在神经细胞缺血损伤中的表达增加以及在神经元的表达定位，已构建TRPM7和DREAM的质粒以及它们的过表达，但二者的相互作用验证的工作正在进行。综上，本研究证实了抑制TRPM7可减轻脑缺血损伤并改善神经功能障碍，同时认识到TRPM7在脑缺血再灌注损伤的急性期的表达高峰的时间段，为研究靶向抑制TRPM7治疗脑缺血损伤提供可能的治疗依据和启示性线索