磷酸化nNOS/Sox2相互作用调控Shh/Gli1信号通路对脑缺血损伤后神经干细胞的影响及其机制

The proliferation and differentiation of neural stem cells (NSCs) is an important way for neural regeneration after cerebral injury. Previous research has found that nNOS can regulate NSCs proliferation and plays an important role during cerebral injury. Our earlier experiment results firstly showed that phosphorylated nNOS (p-nNOS) could enter the nucleus of neuron and form interact with Sox2, then regulate the transcription of Shh after the cerebral ischemia injury. Meanwhile we also found that p-nNOS and Sox2 expressed in the nucleus of NSCs, and p-nNOS interacted with Sox2. Based on these findings above, we hypothesize that the NSCs proliferation and differentiation after cerebral injury may relate to p-nNOS/Sox2 interaction. However, the mechanism of p-nNOS/Sox2 interaction and the relationship between the p-nNOS/Sox2 and Shh/Gli1 signal pathway are still unclear. In this subject, we will firstly investigate the expression and subcellular localization of p-nNOS and its interaction with Sox2 after cerebral ischemia injury, analysis the effect on the phosphorylation of Sox2 as well as the transcription activity of Sox2, and then elucidate the possibility of the changes of NSCs through Shh/Gli1 signal pathway regulated by p-nNOS/Sox2. Finally we will intervene the interaction of p-nNOS-Sox2 to study the neuroprotective role of the p-nNOS/Sox2 during cerebral ischemia injury. This research will clarify the mechanisms of NSCs regulation by p-nNOS/Sox2 interaction and provide both the theoretical and experimental bases to explore the protective mechanisms of NSCs after cerebral ischemia.

神经干细胞(NSCs)增殖、分化是脑损伤后神经再生的重要方式。研究证实nNOS不仅可调节NSCs增殖，同时在脑损伤中起着重要的作用。我们前期发现神经元缺血损伤后磷酸化nNOS（p-nNOS）入核与Sox2相互作用，并调节Shh/Gli1信号通路中Shh的转录；同时在NSCs中我们发现了nNOS/Sox2共定位于胞核，且NSCs损伤后胞核中nNOS以磷酸化状态为主，且p-nNOS与Sox2存在相互作用。由此我们设想脑损伤后NSCs中nNOS磷酸化，p-nNOS/Sox2相互作用并通过调控Shh/Gli1通路来影响NSCs。本课题拟研究脑缺血损伤后NSCs中nNOS磷酸化水平变化；p-nNOS/Sox2相互作用及对Sox2磷酸化的影响；并进而观察p-Sox2通过调控Shh/Gli1通路对损伤后NSCs的影响，从而为脑损伤通过干预NSCs中p-nNOS/Sox2实现神经再生提供理论和实验依据。

通过移植外源性NSCs或激活自身内源性NSCs进行脑损伤后细胞替代治疗成为目前国内外脑损伤治疗的研究热点，而利用NSCs进行脑损伤神经修依然复面临诸多难题，其中最为关键的是移植的NSCs或者内源性的NSCs实际发挥神经再生作用的效率问题，尤其是NSCs在损伤状态下增殖、分化调节的具体分子机制，仍然有待于进一步研究明确，因此阐明这一机制是脑损伤后发挥NSCs神经再生修复作用的关键所在。.鉴于此，本研究发现TBI后NSCs中nNOS在胞核和胞浆中均有表达，NSCs损伤后胞核中nNOS发生磷酸化修饰，且是以ser847位点发生磷酸化为主（即NP847）；同时发现NSCs中NP847与Sox2存在相互作用，并调节TBI后NSCs中Sox2转录活性，进而调控Shh的转录，最终通过Shh/Gli1信号通路来影响NSCs自我更新及增殖，发挥NSCs神经再生修复的作用，从而验证脑损伤后p-nNOS可以与Sox2相互作用来调节NSCs的生物学效应。.除此以外，我们还发现泛素-蛋白酶体系统（UPS）对于NSCs的增殖与分化有着较大的影响。我们发现E3泛素连接酶Triad1可以通过与其相关的其他E1、E2泛素连接酶，来影响Disc1的表达情况，从而最终影响NSCs的增殖与分化。.综上，本项目首次发现nNOS及Sox2是可以促进脑损伤后内源性NSCs动员增殖的有效分子靶点，并明确了其中的分子机制，以及E3泛素连接酶Triad1可以通过与其相关的其他E1、E2泛素连接酶，来影响Disc1的表达情况，从而最终影响NSCs的增殖与分化。相关研究内容国内外未见报道，具有明确的原创性，为TBI后通过内源性NSCs动员实现自身神经修复奠定了实验基础。