糖基化终末产物受体在蛛网膜下腔出血早期脑损伤中的作用研究

发布时间：2018-03-22 22:18

  本文选题：糖基化终末产物受体　切入点：蛛网膜下腔出血　出处：《南京大学》2015年博士论文　论文类型：学位论文

【摘要】：背景:蛛网膜下腔出血(subarachnoid hemorrhage,SAH)是高致死率和致残率的中枢神经系统血管性疾病。大量研究证实早期脑损伤(early brain injury,EBI)是造成SAH患者预后不良的主要原因。EBI的机制复杂,包括血肿机械损伤、脑灌注压下降、血液成分毒性作用、脑水肿、氧化应激、炎性反应、神经细胞凋亡坏死等。临床治疗中通过单一机制干预早期脑损伤无法取得满意治疗效果。因此寻找在SAH后可同时调控多种损伤机制的因子或因素显得愈发重要。国际最新研究表明糖基化终末产物受体(receptor for advanced glycation end-product,RAGE)可同时介导炎性反应,调控细胞凋亡与自噬,参与神经修复。同时,RAGE的多种配体被证实参与SAH后脑损伤的病理生理过程。我们推测RAGE在SAH后早期脑损伤的发生发展中起到一定的作用。目前文献中尚无关于RAGE在SAH后早期脑损伤中作用的报道。因此,本课题针对RAGE在SAH后的表达时程及其在早期脑损伤中的潜在作用机制进行研究。方法:本课题分别建立SAH体内及体外模型,首先运用Western blotting、real-time PCR、免疫组织化学染色及免疫荧光技术在SAH后不同时间点观察RAGE表达水平的变化及其细胞定位。然后根据体内体外实验中RAGE的表达情况,使用其特异性抑制剂FPS-ZM1针对RAGE进行下调,运用Western blotting、酶联免疫吸附实验(ELISA)、免疫组化及免疫荧光技术检测核因子-κB(NF-κB)、TNF-α及IL-1β等炎性因子的表达变化及小胶质细胞的活化水平变化来明确RAGE在SAH后炎性反应中的作用及机制。然后,采用Flouro-JadeB、Nissl染色及流式细胞术分别从体内体外模型中观察下调RAGE后神经细胞损伤情况。为明确RAGE在SAH后神经损伤中的具体作用机制,我们进一步采用Western blotting、免疫荧光技术检测凋亡相关因子(caspase-3、Bcl-2、Bax)及自噬相关因子(LC3、beclin-1)的表达变化。最后,为明确下调RAGE对大鼠SAH后神经功能的影响,我们检测了大鼠脑组织含水量及神经功能评分的变化。结果:体内及体外实验结果均表明:RAGE在SAH后表达水平均较对照组明显增高,同时RAGE的表达见于神经元及小胶质细胞中。SAH后抑制RAGE可显著降低NF-κB及其下游炎性因子TNF-α和IL-1β的蛋白水平,并显著抑制小胶质细胞的活化。提示RAGE可能通过激活NF-κB及调控小胶质细胞介导SAH后的炎性反应。进一步研究发现,使用特异性抑制剂下调RAGE的表达后神经细胞损伤加重,抑制RAGE引起神经细胞凋亡增加,自噬减少。最后,体内实验发现抑制RAGE可显著减轻SAH后1天大鼠脑水肿,改善神经功能,但不能改善SAH后3天大鼠脑组织水肿及神经功能。结论:本课题通过体内、体外实验证实:SAH后RAGE的表达水平显著增高,并在SAH后早期脑损伤中发挥"双向作用",一方面通过NF-kB促进炎性反应引起脑损伤,另一方面通过调控凋亡与自噬减少神经细胞死亡起到脑保护作用。本课题的发现有助于进一步了解SAH后早期脑损伤的具体机制,也提示通过干预RAGE来治疗SAH可能存在潜在的局限性。
[Abstract]:Background: subarachnoid hemorrhage (subarachnoid hemorrhage SAH) is a high mortality rate and disability rate of vascular diseases in central nervous system. Many studies have confirmed that early brain injury (early brain, injury, EBI) is the main reason causing mechanism of.EBI SAH patients with poor prognosis of the complex, including hematoma mechanical damage, cerebral perfusion pressure decreased blood components, toxicity, brain edema, oxidative stress, inflammatory reaction, apoptosis and necrosis. The clinical treatment by single intervention mechanism of early brain injury can not be achieved satisfactory results. So looking at SAH after injury and the regulation mechanism of various factors or factors become increasingly important. The latest research shows that the international sugar group end product receptor (receptor for advanced glycation end-product, RAGE) can mediate inflammatory reaction, cell apoptosis and autophagy, involved in nerve repair. At the same time, a variety of RAGE The ligand proved to be involved in the pathophysiological process of brain injury after SAH. We speculated that RAGE in early brain injury after SAH play an important role in the development of the literature. There is no report about RAGE in early brain injury after SAH effect. Therefore, the topic for the RAGE in the SAH after the expression of history and Study on the potential role in mechanisms of early brain injury. Methods: SAH in vivo and in vitro models are established in this paper, we first use the Western blotting, real-time PCR, immunohistochemical staining and immunofluorescence technique in SAH at different time after the observation of the change of the expression of RAGE and its cellular localization. Then according to the expression of RAGE in vivo and in vitro the use of its inhibitor FPS-ZM1 for RAGE reduction, using Western blotting, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry and immunofluorescence detection of nuclear factor Sub - kappa B (NF- K B), activated expression of TNF- alpha and IL-1 beta and other inflammatory factors and microglia to clear level changes and mechanism of RAGE in inflammatory reaction after SAH. Then, using Flouro-JadeB, Nissl staining and observe respectively the neuronal damage after down-regulation of RAGE in vivo in vitro model of medium flow cytometry. To determine the RAGE in specific mechanism of nerve injury in SAH, we used Western blotting, immunofluorescence detection of apoptosis related factors (Caspase-3, Bcl-2, Bax) and autophagy related factors (LC3, beclin-1) expression changes. Finally, to clear the neurological effects of down-regulation of RAGE function after SAH in rats, we examined the changes of brain water content and neurological score. Results: in vivo and in vitro experimental results show that the expression of RAGE in SAH were higher than control group, while the expression of RAGE. 浜庣�炵粡鍏冨強灏忚兌璐ㄧ粏鑳炰福�

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