双孔钾通道TREK-1在星形胶质细胞-神经元间谷氨酸代谢中的作用

The newly found tandem-pore potassium channel TREK-1, have characters of concentrated central distribution and strongly pathological activation, is the new target for cerebral ischemia therapy. However, the regulation mechanisms of TREK-1 on cerebral ischemia is still unkown. The applicant have discovered that TREK-1 protein highly expressed in astrocytes, and affected their ability to glutamate uptake. Excessive glutamate induced excitatory neural toxicity is the most important factor in neurons death during cerebral ischemia. Accordingly, the applicant proposed that TREK-1 can affect neurons survival state through regulating the glutamate cycle mediated by astrocytes after brain ischemia. On the basis of glutamate metabolic regulation, using RNA transfection technology to over-express astrocytic TREK-1, combined with astrocytes-neurons co-culture and cerebral ischemia in vitro methods, the project will systematically investigate the influence of TREK-1 on interaction between different state (healthy/ischemic injury) astrocytes (wild type/TREK-1 over-express type) and ischemic neurons by using various dectecion means including patch clamp, RT-PCR and Western, to clear the function and mechanism of TREK-1 on neurons injury in the process of cerebral ischemia, and provide the theory basis for the treatment of ischemic diseases.

新型双孔钾通道TREK-1，具有中枢集中分布和脑缺血等病理性强烈激活的特点，是脑缺血治疗的新靶点，但其对脑缺血的调节机制尚不明确。申请者已发现TREK-1蛋白高表达于星形胶质细胞，影响其谷氨酸摄取能力，而谷氨酸兴奋性毒性是脑缺血引起神经元死亡的重要因素。据此，申请者推测脑缺血时TREK-1可通过调节星形胶质细胞谷氨酸代谢功能，影响突触间隙谷氨酸水平，而对神经元产生作用。故项目拟用过表达TREK-1星形胶质细胞、共培养和体外脑缺血方法，以谷氨酸代谢为核心，应用膜片钳、RT-PCR、Western等检测手段系统考察TREK-1对不同状态(正常/缺血损伤)星形胶质细胞（野生型/ TREK-1过表达型）与缺血神经元相互作用的影响，明确TREK-1在脑缺血所致神经元损伤过程中的作用及机制，为脑缺血疾病的治疗提供理论依据。

研究表明新型双孔钾通道 TREK-1在人和动物大脑中枢呈集中性分布，在脑缺血、缺氧的病理性情况下可强烈激活，是脑缺血治疗的可能靶点，但TREK-1通道对脑缺血神经元和胶质细胞的影响及调节机制尚不明确。我们发现 TREK-1 蛋白高表达于星形胶质细胞，影响其谷氨酸摄取能力，而谷氨酸兴奋性毒性是脑缺血引起神经元死亡的重要因素。所以，申请者推测脑缺血时 TREK-1 可通过调节星形胶质细胞谷氨酸代谢功能，影响突触间隙谷氨酸水平，而对神经元产生作用。项目用过表达 TREK-1 星形胶质细胞、共培养和体外脑缺血方法，以谷氨酸代谢为核心，应用RT-PCR、Western等检测手段系统考察 TREK-1 对不同状态(正常/缺血损伤)星形胶质细胞（野生型/ TREK-1过表达型）与缺血神经元相互作用的影响，结果提示：星形胶质细胞的双孔钾通道TREK-1表达水平可影响星形胶质细胞的谷氨酸代谢功能，而且缺氧与否，影响不同；星形胶质细胞与神经元共培养时，星形胶质细胞的双孔钾通道TREK-1表达水平和缺氧与否对神经元的存活影响不同，神经元的状态不同对星形胶质细胞（野生型/ TREK-1过表达型）的谷氨酸代谢功能影响不同。证实了项目假说：脑缺血时，双孔钾通道TREK-1 通道可调节星形胶质细胞谷氨酸代谢功能，调节突触间隙谷氨酸水平，而决定神经元的生存状态，双孔钾通道TREK-1可作为脑缺血治疗的新靶点。