TMEM166基因在小鼠脑缺血再灌注损伤中的功能研究

Stroke and its sequelae continue to be a leading cause of disability and mortality worldwide. Despite interventional treatments that significantly decrease the general incidence of stroke, a majority of patients suffer from serious ischemia/reperfusion injury in the process. Our previous study showing TMEM166’s involvement in neuronal excessive autophagy and the subsequent brain damage following ischemia/reperfusion in rats suggests TMEM166 to be a potential novel therapeutic in human stroke. However, the underlying mechanism is unclear and TMEM166’s role in this process requires further cautionary evaluation. In this proposal, we will explore the role of TMEM166 after ischemia/reperfusion brain injury with TMEM166 knock-out mice and focus on the possible neuronal autophagy mechanisms regulated by TMEM166. .Studies have proposed that AMPK-mTOR and Becklin-1 are important regulators of autophagy in response to ischemia/reperfusion injury. Our preliminary results suggest ischemia activates AMPK and inhibits mTOR while reperfusion markedly increases Beclin-1 and rapidly inactivates AMPK. Both AMPK and Beclin-1 expression were inhibited in TMEM166 knock-out animals. Thus, we hypothesized that TMEM166 regulates neuronal autophagy through AMPK-mTOR related mechanisms during ischemia and Beclin-1 during reperfusion. The program contains two primary goals: investigate the role of TMEM166 in a rodent ischemia/reperfusion model and examine the mechanism by which TMEM166 regulates autophagy during ischemia/reperfusion with Compount C (AMPK-specific inhibitor), TSC2shRNA, Beclin-1 siRNA, and adenovirus expression vector (Ad5- TMEM166).

脑中风是导致全球病人致死和致残的重要原因。我们研究发现TMEM166与脑缺血损伤后的神经元自噬及由此导致的神经损伤密切相关。本研究在TMEM166基因敲除鼠脑缺血再灌注损伤模型(MCAO)的基础上观察其作用并重点探讨其机制。.AMPK-mTOR和 Beclin-1是脑缺血后自噬的重要调节因子。我们的预实验证实在脑缺血阶段AMPK-mTOR被激活，IR阶段AMPK失活Beclin-1升高，TMEM166基因敲除鼠AMPK及 Beclin-1的表达均被抑制。由此我们假想TMEM166分别通过AMPK-mTOR或Beclin-1实现其对MCAO后自噬的调控。本实验从两方面验证假说（1）观察TMEM166诱导的自噬分别在缺血和IR损伤中发挥的作用；（2）采用Compound C、TSC2shRNA、Beclin-1 siRNA及Ad5-TMEM166深入探讨TMEM166作用机制及临床应用前景。

中风及其后遗症仍是全球范围内致死致残的首要原因，虽然目前中风的发生率已显著下降，但仍有很多人在治疗过程中发生严重的缺血再关注损伤。TMEM166基因定位于人2号染色体短臂12区(2p12)，是最近通过高通量筛选首次证实与程序性细胞死亡相关的人类新基因。研究发现，过表达TMEM166 可以抑制HeLa 细胞的克隆形成能力，最终引起 HeLa 细胞的凋亡及自噬 性死亡。本项目采用线栓法制作MCAO模型，拟观察脑缺血再灌注损伤模型后TMEM166的表达变化，探讨其与脑缺血损伤后神经细胞凋亡及自噬之间的关系及可能机制。项目中我们采用SPF级9-10周龄C57/BL6J雄性小鼠50只，采用随机数字表法分为缺血90 min再灌6、12、24及48 h组，每组12只。通过免疫组化染色观察缺血侧大脑皮层及基底核 TMEM166、Cleaved Caspase 3及 LC3-II 的阳性细胞数量变化，Western blot 检测不同再灌注时间 TMEM166、Cleaved Caspase 3及LC3-II 蛋白水平的表达。缺血侧TMEM166的表达随着再灌注时间的延长而增加，24 h达到高峰，48 h后逐渐下降；再灌注6 h后Cleaved Caspase 3及LC3-II观察到有表达，同样随着再灌注时间而升高，24 h达到高峰。缺血侧Cleaved Caspase 3及LC3-II阳性细胞数量的变化趋势与TMEM166基本一致，对侧大脑半球上述指标无明显变化。因此我们得到初步结论小鼠局部脑缺血再灌注损伤可以诱导 TMEM166的表达，通过激活Caspase 3及LC3-II引起神经细胞凋亡与自噬。