【作者】 夏玉叶；

【导师】 刘全海；

【作者基本信息】 上海医药工业研究院 ， 药理学， 2006， 博士

【摘要】 中枢神经系统的炎症反应，在缺血性脑损伤的病理生理过程中发挥着重要的作用，尤其是在缺血后的继发性损伤及损伤的扩大的过程中扮演重要角色。缺血脑组织中的炎症因子的表达与脑缺血发生、发展和愈后有密切联系。脑缺血损伤后在受损区有大量细胞因子的表达和炎细胞的浸润，可加重脑组织损伤，尤其在再灌注中更加明显。在这些炎症因子中，白细胞介素(IL)是最重要的因子之一。各种IL均可参与炎症反应，并发挥着不同的作用。IL-1β是一个调控急性脑缺血复杂病理变化的抗炎因子，脑缺血及再灌后IL-1βmRNA蛋白水平升高，在大鼠大脑中动脉阻断模型上，缺血15分钟既有IL-1βmRNA的明显表达，3h达到高峰，4天内逐渐消失，表明其在局部脑缺血性炎症中起了重要作用。IL-1β与缺血性、兴奋性和外伤性脑损伤均有关。体内还存在天然的IL-1受体拮抗剂(IL-1ra)，IL-1ra是分子量为23～25kD的糖苷化的蛋白，是IL-1活性的天然产生的抑制物，与IL-1竞争结合IL-1受体而不产生IL-1的信号转导。 Anakinra为基因重组的、非糖基化的人IL-1受体拮抗剂(rhIL-1ra)，其氨基酸序列除N-末端增加甲硫氨酸残基外与人天然IL-1受体拮抗剂一致，153个氨基酸组成，分子量17.3kD，由E.coli表达，已被批准在临床用于治疗类风湿性关节炎。在正常动物其不能通过血脑屏障。将Anakinra静脉注射用于抗脑缺血治疗的动物实验研究和脑微血管内皮细胞的影响尚无报道。我们应用多种脑缺血模型研究Anakinra对脑缺血的治疗作用及治疗时间窗，并从分子水平研究其抗脑缺血的作用机制。可为探索通过拮抗炎症反应的机制来治疗缺血性脑损伤提供依据，寻找具有更长时间窗的脑缺血治疗方法，同时研究在脑缺血时血脑屏障系统对生物大分子的通透情况，这将为开辟新的脑缺血损伤的治疗途径提供更加科学、客观、现代化的手段和方法。 在本研究中，我们采用永久性或缺血再灌注性MCAO模型造成脑缺血性损伤，在缺血后3、6和12小时通过静脉注射Anakinra，发现可明显减少神经功能和脑组织的损害，提示Anakinra在脑缺血时可通过血脑屏障，产生治疗作用。在缺血性脑损伤的治疗上，以往药物的治疗时间窗均较短。当脑缺血时，随时间延长半暗区逐步转化为梗死区，这种转化可能在时间轴上存在不同的机制。从炎症反应的时间关系上提示在梗死的后期进展中，炎性细胞因子表达增加及炎症的发展对脑组织的损伤进展逐渐趋于占据主导地位。本研究中发现，在缺血后12小时给于Anakinra，仍有明显的治疗缺血性脑损伤的作用，其对缺血性脑损伤的治疗时间窗较长，提示具有这一可能的作用机制。 在缺血引起的脑组织损伤上，炎性细胞因子可能扮演了重要的角色，并介导了神经细胞的凋亡和坏死，同时也有文献报道兴奋性氨基酸的兴奋毒性也可能促使IL-1的释放。在本研究中，我们发现在脑缺血区存在大量的凋亡细胞，也证实缺血引起的脑损伤除坏死外，凋亡等也是机制之一。静脉注射Anakinra可减少缺血引起的脑梗死体积，同时神经细胞凋亡也明显减少，Anakinra通过拮抗IL-1的作用对缺血后神经细胞凋亡产生抑制作用，从而增强了其抗脑梗死的药效。在本研究中，Anakinra能拮抗谷氨酸致体外培养神经元细胞的更多还原

【Abstract】 Inflammatory reactions of central nerve system play an essential role in the pathophysiologic process of ischemic brain injury, especially during the enlargement of damage and the secondary damage after ischemia. Proinflammatory factor expression in ischemic brain has a close relationship with occurrence, development and prognosis of cerebral ischemia. There are huge cytokine expression and proinflammatory infiltration in damage area after ischemic brain injury, which aggravate brain injury especially in reperfusion. Interleukin (IL) is one of the most important factors among these proinflammatory factors. All kinds of interleukin take part in inflammatory reactions and play various effects. IL-1β is an inflammatory factor regulating the complex pathologic changes of acute cerebral ischemia. IL-1β mRNA protein level increases after cerebral ischemia and reperfusion. In a model of middle cerebral artery occlusion, IL-1β mRNA was expressed markedly at 15 min after ischemia, peaked at 3 hr and died away during four days suggesting IL-1β plays an important role in inflammation of local cerebral ischemia. IL-1β is relative with ischemic, excitatory and traumatic brain injury. There is a native interleukin-1 receptor antagonist (IL-1ra) in the body which is a glycosylated protein with 23～25 kDa molecular weight and a native inhibitor of IL-1 receptor antagonist (IL-1ra). IL-1ra competitively inhibits the binding of IL-1 to the IL-1R and produces no IL-1 signal transduction.Anakinra, a recombinant, non-glycosylated form of human interleukin-1 receptor antagonist (rhIL-1ra), is identical to a native IL-1 receptor antagonist except for a methionine residue that has been added to the beginning of the amino acid sequence. It is expressed in E. coli, form of 153 amino acids and the molecular weight is 17.3 kDa which is recommended to treat rheumatoid arthritis. It cannot pass through the blood brain barrier in normal animals. There is no article about animal studies of Anakinra being administered iv in cerebral ischemia . We have analysed therapeutic effect and therapeutic time window of Anakinra on cerebral ischemia in several experimental models and its mechanism for therapy of cerebral ischemia at molecular level. This study provides evidence for the treatment of cerebral ischemia by inhibiting inflammatory reaction. We also searched therapy for cerebral ischemia with longer time window and investigated permeability of blood brain barrier system for biological macromolecules in cerebral ischemia, which represents a more scientific, more impersonal and more modern approach to the treatment of ischemic brain injury.In this study, we produced ischemic brain injury by permanent or ischemia-reperfusion model of middle cerebral artery occlusion (MCAO). Anakinra was injected intravenously at 3,6,12 hr after ischemia. It was found that Anakinra obviously reduced the damage of nervous function and brain, which indicated Anakinra could cross blood brain barrier for treatment in cerebral ischemia. The therapeutic time window of previous drugs for the treatment of cerebral更多还原