受体相互作用蛋白3（Receptor-interacting protein 3，RIP3）调控神经元缺血性程序性坏死的作用及机制研究

The studies about programmed necrosis made great progress recently, receptor-interacting protein 3 (RIP3) was found to be the molecular switch of programmed necrosis. Our recent studies showed that the changes of hippocampal cornu ammonis 1 neuronal death induced by global cerebral ischemia/reperfusion injury were programmed necrosis. And our following studies showed that the expression of RIP3 after ischemia/reperfusion injury increased greatly in neuronal nuclei, which has not been reported and was different with programmed necrosis induced by tumor necrosis factor. These suggested RIP3 may be the key regulator of neuronal programmed necrosis after global cerebral ischemia/reperfusion injury, and the regulated target may be in the nuclei. In this project, from the whole animal and cellular level, we try to study the changes of RIP3, and the effect of increased expression and nuclear location of RIP3 on neuronal death; and try to clarify the downstream signaling pathway of neuronal programmed necrosis regulated by RIP3. Molecular biology techniques and tools of RIP3 knock-out mouse, RNA interfering, gene transfection and expression, drug inhibition, co-immunoprecipitation, double immunofluorescent labeling and electron microscopy would be employed in this study. Implementation of this project is help to reveal the role and signal transduction mechanisms of RIP3 in neuronal ischemic programmed necrosis. This would provide a theoretical reference for cerebrovascular disease research, and find effective intervention target for cerebrovascular disease treatment.

近年来程序性坏死研究取得重要进展，受体相互作用蛋白3（Receptor-interacting protein 3，RIP3）被发现是程序性坏死的分子开关。本课题组前期研究发现全脑缺血复灌损伤后神经元死亡特征符合程序性坏死，随之我们发现缺血损伤诱导RIP3在神经元核内表达显著升高，有别于肿瘤坏死因子诱导的程序性坏死，提示RIP3可能是调控神经元缺血性程序性坏死的关键分子，且靶位在细胞核内，该结果尚未见报道。本项目拟利用基因敲除小鼠，应用RNA干扰、基因转染、拮抗剂干预等方法；采用免疫共沉淀、免疫荧光双标、电镜等技术；从整体动物和细胞水平研究神经元缺血性程序性坏死时RIP3的变化特征及RIP3表达增高、核定位对神经元死亡的影响，阐明RIP3调控神经元程序性坏死的下游信号通路。该项目的实施有助于揭示RIP3在神经元缺血性程序性坏死中的作用及调控机制，有望为脑血管病治疗寻找新的有效干预靶点。

我们课题组前期研究发现全脑缺血/复灌损伤诱导的大鼠海马CA1区神经元死亡特点符合程序性坏死，而非传统的凋亡模式，众多研究表明受体相互作用蛋白3（Receptor-interacting protein 3，RIP3）是程序性坏死的关键调节因子。在这个研究中，我们主要探讨RIP3是否为介导神经元缺血性程序性坏死的关键分子以及RIP3调控神经元程序性坏死的下游信号分子和信号通路。通过一系列的实验研究，我们发现：（1）RIP3是程序性坏死的关键调节因子，在缺血/复灌损伤下其介导神经元死亡的关键一环是移位入细胞核，用不同类型的抑制剂阻止RIP3的核位移可以防止神经元死亡；（2）采用程序性坏死特异性抑制剂Nec-1、自噬抑制剂3-MA和caspase-3抑制剂Ac-DMQD-CHO预处理干预全脑缺血/复灌损伤，发现Nec-1和3-MA对全脑缺血/复灌损伤后的神经元死亡有保护作用，而Ac-DMQD-CHO不能抑制海马CA1区神经元死亡，并且caspase-3在此过程中未见激活，caspase-8主要表达于胶质细胞而在神经元中未见明显表达，进而说明caspases不参与神经元缺血性程序性坏死；（3）进一步研究发现RIP3与AIF在细胞核内共定位并且相互结合形成复合体是缺血/复灌损伤诱导神经元程序性坏死的关键，Nec-1和3-MA能够抑制二者的结合从而减轻全脑缺血/复灌损伤引起的神经元死亡。综合本课题及我们前期的课题研究结果，我们得出如下结论：①缺血/复灌损伤后的神经元死亡为程序性坏死，caspases不参与此过程；②RIP3、AIF转移入核并且相互结合形成复合体是此过程的关键；③程序性坏死抑制剂Nec-1及自噬抑制剂3-MA对此有抑制作用且对神经元死亡有保护作用，说明3-MA不仅抑制自噬，也参与调节程序性坏死过程。