RIP1/RIP3在脑缺血损伤“程序性坏死”中的作用机制研究

Cerebral ischemia with extremely high morbidity rate lacks effective treatment. Recent studies have found that a controllable way of cell death "Necroptosis" exists in ischemic cerebral damage. But its mechanism is not thoroughly clarified. Necrosome which consists of receptor-interacting protein 1 and 3 (RIP1, RIP3) promotes the initiation of programmed cell death. Our group has confirmed that RIP1 promotes Necroptosis after cerebral ischemic damage happened, however, recent researches strongly indicate that RIP1 exhibits its Necroptosis-inducing function only when RIP3 exists. RIP3 promotes Necroptosis by accelerating glycolysis, and this process is related to the increased density of methylglyoxal (MG) and advanced glycosylation end products (AGEs). So, the main role of RIP3 is accelerating glycolysis to provide energy to damaged cells after cerebral ischemia, but the over-activation of RIP3 may inhibit the α-oxoaldehyde metabolic detoxification system, which results in the accumulation of MG and AGEs to induce the cytotoxity. RIP3 may be mostly pivotal regulator of the cerebral ischemic Necroptosis. In the present study we try to manifest the role of RIP3 in Necroptosis after cerebral ischemia, by inhibiting the activity of RIP1 and interfering the RIP3 expression in vivo and vitro. RIP3 downstream signaling pathways are also planned to be preliminarily studied.

高残死率的缺血性脑卒中缺乏有效治疗手段，近来新发现一种可调控的细胞死亡方式程序性坏死在脑缺血损伤中存在，具体调控机制不详。受体相互作用蛋白1和3(RIP1，RIP3)组成促坏死复合体促进程序性坏死的发生，本课题组已证实RIP1参与脑缺血损伤后程序性坏死的调控。但新近研究强烈提示：RIP1只有在RIP3存在的条件下才能诱导程序性坏死的发生，RIP3加速糖酵解促进程序性坏死发生的同时检测到甲基乙二醛(MG)和晚期糖基化终产物(AGEs)浓度增高。因此，脑缺血损伤后RIP3的主要作用是加速糖酵解为受损细胞提供能量，但RIP3的过度激活可能抑制MG等α-羰基醛的代谢解毒系统使MG和AGEs积聚发挥细胞毒性，RIP3可能是脑缺血损伤后程序性坏死最关键调控分子。拟抑制RIP1的活性和体内外靶向干扰RIP3的表达进一步明确RIP3在脑缺血损伤后介导程序性坏死中的作用，并初步探索RIP3的下游信号机制。

2005年Degterev等提出一种新的细胞死亡方式：细胞程序性坏死。细胞的这种死亡方式具有坏死样结构特点，细胞膜完整性破坏，周围组织炎症反应，同时该过程受细胞内一系列信号分子调控。脑缺血所致脑损伤是我国神经科致死、致残的主要病因之一。如果能通过深入探索程序性坏死信号通路的生物化学机制，更进一步研究程序性坏死信号通路在相关疾病中的参与程度与作用，不但利于对该信号通路的认知，且对神经内外科相关神经缺血再灌注损伤相关疾病的治疗药物、治疗靶向、治疗方法起到拓宽、确立的重要作用。.[目的] 本实验研究体外培养神经元细胞在缺血再灌注损伤后RIP3的变化规律。本实验受临床上脑梗死后出现缺血半暗带启发，研究并探讨程序性坏死在半暗带区和梗死区的分布，进一步揭示程序性坏死在脑缺血再灌注损伤中的作用机制。.[方法] 原代神经元培养，神经元细胞荧光鉴定，RIP3荧光鉴定，传代细胞HT22细胞培养及冻存，细胞造缺氧模型，动物造缺氧再灌注模型，PCR技术，Western Blotting技术，MTT技术，鼠脑TTC染色.[结果] RIP3表达在缺血再灌注在体和体外模型中都有显著变化。在本实验所设之间点中，在缺血再灌注损伤后12小时RIP3表达达到高峰。小鼠海马来源细胞系HT22与大鼠皮层神经元造模后PCR结果趋势一致。动物造模后梗死区及半暗带区Western Blotting趋势一致，与细胞PCR趋势一致。在体实验中，梗死区较半暗带区，RIP3表达波动显著。缺氧时间1小时NEC-1实验组细胞存活率较对照组升高不明显，而缺氧时间为4小时NEC-1实验组细胞存活率较对照组显著升高。通过慢病毒（抑制RIP3表达）局部转染大鼠脑组织，并造大鼠脑缺血再灌注模型证实抑制RIP3表达能减轻大鼠脑组织缺血再灌注损伤..[结论] 脑缺血再灌注模型中，缺血区和半暗带区由于损伤程度不同，程序性坏死参与度不同，梗死区程序性坏死更活跃。