Ykt6与SEC22b在脑缺血再灌注后神经元自噬流中的调控作用及机制研究

Neuronal injury is an important part of cerebral ischemia-reperfusion (I/R) injury. Our preliminary experimental results showed that I/R induced autophagic flux damage and that Ykt6 and SEC22b were probably involved in the process. Thus, we proposed the hypothesis: Ykt6 and SEC22b mediate the integrity of autophagic flux in neurons. After I/R, the expression of Ykt6 and SEC22b is disturbed, which promotes autophagic flux damage, and finally brain injury. To test the hypothesis, we use experimental I/R model in rats. First, we discover the effect of I/R on autophagic flux and the expression of Ykt6 and SEC22b in neurons of the penumbral tissue. Second, gene transfection, RNAi and transgenic mice are used to regulate the target gene expression, and then to evaluate the role of Ykt6 and SEC22b in I/R-induced autophagic flux damage and brain injury. Third, autophagy-blocking agent and autophagy-deficient mice are used to test whether Ykt6 and SEC22b exert their effects via regulating autophagy. And finally, by double immunofluorescence, we demonstrate the mechanisms underlying the effect of Ykt6 and SEC22b on I/R-induced autophagic flux damage and brain injury. The longitudinal studies from three levels of cell, tissue and in vivo, horizontal researches from three levels of ultrastructure, molecular biology and behavior in this study will provide us new ideas and theoretical basis to improve the prognosis of ischemic stroke patients.

神经损伤是脑缺血再灌注（I/R）损伤的重要组成部分。我们预实验结果提示: I/R后神经元自噬流破坏，Ykt6与SEC22b可能参与其中。为此，我们提出假说：Ykt6与SEC22b参与神经元自噬流，I/R后二者表达失衡，导致神经元自噬流紊乱，加剧脑损伤。为了验证该假说，我们拟利用大鼠I/R模型，应用电镜和western blot等检测神经元自噬流以及Ykt6与SEC22b的水平；应用基因调控和转基因，评估调控Ykt6与SEC22b表达的关键因子以及二者的表达变化对神经元自噬流完整性和神经损伤的影响；利用自噬流抑制剂及自噬缺失小鼠阻断自噬，明确Ykt6与SEC22b是否通过调控自噬流发挥作用；利用免疫荧光染色等技术，明确Ykt6与SEC22b调控神经元自噬流的机制。本研究纵向从细胞、组织和活体三个层面，横向从超微结构，分子生物学和行为学三个层面研究，将为改善脑I/R 预后提供新思路和理论依据。

缺血半暗区脑缺血-再灌注损伤是缺血性脑卒中患者再通治疗效果不佳的重要原因。有充分的证据表明自噬在缺血性脑卒中患者脑损伤中具有双重作用。本研究旨在了解缺血半暗区自噬的特点及自噬双重作用的可能机制。我们发现，在缺血前，雷帕霉素和碳酸锂诱导自噬可减少神经功能缺损和梗死，而在雄性小鼠大脑中动脉闭塞/再灌注模型中，诱导再灌注后的自噬作用相反，这两种作用在自噬缺失小鼠中均被消除(Atg7flox/flox;nestin-Cre)。自噬流测定表明，再灌注导致神经元轴突自噬小体逆行转运受阻，进而导致自噬流损伤。然后，我们发现缺血再灌注诱导神经元中Sec22b和Ykt6这两个自噬小体转运相关因子的蛋白水平发生变化，其中Sec22b显著升高，Ykt6显著降低。Sec22b敲低和Ykt6过表达以自噬依赖的方式显著减轻脑缺血再灌注神经元或小鼠的自噬流障碍、梗死和神经功能缺损。此外，Sec22b基因敲低和Ykt6过表达将雷帕霉素治疗后的预后从恶化转变为神经保护。因此，Sec22b和Ykt6在神经元自噬流中发挥关键作用，适当调节Sec22b和Ykt6可能有助于逆转靶向自噬诱导的失败，改善缺血性卒中的预后。