基质金属蛋白酶9经水通道蛋白4参与缺血性脑水肿形成的机制研究

The peak of ischemic brain edema often occurred in the 24 hours after the onset of ischemic stroke, unfortunately, it is lack of the effective drugs clinically preventing the formation of brain edema. Aquaporin-4(AQP4) is the key factor of ischemic brain edema. Studies have shown that metalloproteinase 9 (MMP-9) plays an important role in the injury of blood-brain barrier and secondary brain edema after cerebral ischemia. In the animal models of traumatic brain injury, subarachnoid hemorrhage or herpes simplex encephalitis, when the MMP-9 expression was inhibited, AQP4 expression was along with the inhibition, suggesting that MMP-9 may be involved in the regulation of AQP4 generated, thereby affecting the progress of brain edema. Combined with the previous experimental basis and literature study, we hypothesis that the “MMP-9 may be involved in regulating the generation of AQP4，and further influences the formation of ischemic brain edema”. Is it real？What is the mechanism？Based on this, the cellular or rat models of cerebral ischemia will be establishing, and MMP-9 should be silenced using the siRNA in this project. Then, the water permeability and expression of AQP4 as well as the related regulation mechanism of MMP-9 will be detected by methods of cellular and molecular biology such as Immunohistochemistry, Western blot, membrane water permeability measurement and so on. The expected results will not only elucidate the molecular mechanism of MMP-9 affecting AQP4 in ischemic brain edema，but provide new ideas and target for early prevention of ischemic brain edema.

缺血性脑卒中的脑水肿高峰期多在发病24小时后，然而临床上尚缺乏有效的药物阻止脑水肿高峰期的形成。水通道蛋白4（AQP4）是缺血性脑水肿发生发展中的关键因子。基质金属蛋白酶9（MMP-9）在脑缺血后血脑屏障损伤及继发性脑水肿中起着重要作用。在脑外伤、蛛网膜下腔出血或脑炎等疾病模型中，MMP-9表达被抑制后，AQP4的表达也随着下调，提示MMP-9可能与AQP4变化有关，进而参与影响脑水肿的进展。那么，MMP-9是否对脑缺血的AQP4有调节作用？其作用机制是什么？目前尚未明了。据此，本项目通过建立脑缺血细胞和动物模型，经siRNA沉默MMP-9后，拟运用免疫组化、免疫蛋白印迹、质膜水通透性测定等多种实验方法检测AQP4水通透性和表达的变化及其调控机制。本研究不仅完善MMP-9在缺血性脑水肿发病过程中的作用机理，还为缺血性脑水肿的早期防治提供了新的思路和靶点。

缺血性脑卒中脑水肿高峰期多在发病24小时后，然而临床上尚缺有效的药物阻止脑水肿的形成。水通道蛋白4（AQP4）是缺血性脑水肿发生发展中的关键因子。基质金属蛋白酶9（MMP-9）在脑缺血后血脑屏障损伤及继发性脑水肿中起着重要作用。在蛛网膜下腔出血等疾病模型中，MMP-9表达被抑制后，AQP4的表达也随着下调，提示MMP-9可能与AQP4变化有关，进而参与影响脑水肿的进展。然而，MMP-9是否对脑缺血的AQP4有调节作用，其相关机制未明。本项目通过建立脑缺血动物模型和缺氧缺糖星形胶质细胞模型，分别经siRNA沉默MMP-9后，运用免疫组化、RT-PCR、免疫蛋白印迹、质膜水通透性测定等多种实验方法检测AQP4表达和水通透的变化，并通过检测激酶PKA、PKC、PKG或CaMKII的变化来探索其调控机制。本项目研究结果显示，脑缺血后大鼠大脑的MMP-9和AQP4的表达升高；研究MMP-9对缺血性脑水肿AQP4表达的影响，发现MMP-9有上调缺血性脑水肿AQP4表达的作用，脑缺血会导致血脑屏障通透性增加，而抑制MMP-9或抑制AQP4后可以减轻脑缺血所致的血脑屏障损害；MMP-9通过调控AQP4进而促进脑水肿的形成；MMP-9能通过激活PKC来上调AQP4，进而加重脑水肿。本研究不仅完善MMP-9在缺血性脑水肿发病过程中的作用机理，还为缺血性脑水肿的早期防治提供了新的思路和靶点。