RGMa对大鼠大脑皮质神经元缺血再灌注引发自噬的作用

发布时间：2018-06-08 22:27

  本文选题：RGMa + 6FNIII　； 参考：《重庆医科大学》2017年硕士论文

【摘要】：背景与目的:脑血管病是对人类健康极具威胁的常见病,是目前人类三大致死率最高的疾病之一,而缺血性脑血管病是其主要类型。脑组织缺血将会导致脑组织损害,而脑缺血后再灌注过程往往加重这一损害。自噬(autophagy)是真核细胞中一种高度保守的,形成囊泡并与溶酶体结合后降解长效蛋白、受损蛋白及受损细胞器,为细胞的代谢、发育提供养分和能量,维持细胞内稳态的过程。大量研究发现,脑缺血再灌注可诱发细胞自噬,且自噬过程基本发生于神经元,主要与饥饿、生长因子缺乏、病原体感染等有关,是脑缺血再灌注损伤的重要环节,此过程受一系列复杂的信号分子调控,其中Beclin1分子是自噬调控通路中最主要的分子之一,其与PIK3C3结合形成Beclin1-PIK3C3复合物,广泛参与自噬体的形成与成熟。不少研究者发现,其目的蛋白通过作用于Beclin1-PIK3C3复合物影响自噬过程,此过程称为“Beclin1依赖性自噬”,而部分研究者发现其目的蛋白不通过Beclin1作用影响自噬,此过程称为“Beclin1非依赖性自噬”。近年来有研究发现脑缺血再灌注损伤后可导致的神经元自噬过程,而Beclin1非依赖性自噬与促进凋亡水平、抑制脑功能恢复有关。因此,自噬可能成为未来治疗脑缺血性疾病的重要靶点。排斥导向分子a(Repulsive guidance molecule a,RGMa)是一种排斥性轴突导向分子,大量研究发现,脑缺血再灌注后RGMa表达增多,正是由于RGMa等一系列轴突生长抑制因子的存在,使得中枢神经系统(CNS)损伤后神经元修复及神经功能恢复极为困难。我们之前的研究发现,在大鼠脑缺血损伤后干预RGMa表达,可显著改善缺血再灌注脑损伤轴突的生长情况并有效促进神经功能恢复。6FNIII为RGMa受体neogenin分子中6个纤维蛋白Ⅲ样结构域,外源性6FNIII在体内可以和RGMa分子结合而抑制其生物学作用的发挥。本实验利用6FNIII对RGMa/Neogenin通路进行干预以及重组腺病毒抑制RGMa蛋白表达两种干预手段探讨RGMa对大鼠大脑皮质神经元缺血再灌注急性期引发的自噬以及神经功能恢复情况的影响,同时对RGMa影响自噬的机制作初步探索。方法:1.将30只雄性成年SD大鼠随机分为5组:假手术组(sham)、脑缺血再灌注组(I/R)、脑缺血再灌注加低浓度6FNIII干预组(I/R+L-6FNIII)、脑缺血再灌注加中浓度6FNIII干预组(I/R+M-6FNIII)、脑缺血再灌注加高浓度6FNIII干预组(I/R+H-6FNIII),立体定位侧脑室注射等体积不同浓度6FNⅢ后立即建立大脑中动脉阻塞(MCAO)模型,所有组于再灌注后24h,采用Western-blot检测RGMa下游分子CRMP-2蛋白表达水平,确定最佳6FNIII干预浓度。2.将108只雄性成年SD大鼠随机分成6组:假手术组(sham)、脑缺血再灌注组(I/R)、脑缺血再灌注加生理盐水注射组(I/R+NS)、脑缺血再灌注加6FNIII干预组(I/R+6FNIII)、脑缺血再灌注加空载体腺病毒注射组(I/R+r Ad-HK)及脑缺血再灌注加RGMa重组腺病毒干预组(I/R+r Ad-sh RGMa)。6FNIII干预组及NS注射组于6FNIII或NS侧脑室注射后立即建立MCAO模型,腺病毒干预组于RGMa病毒大脑皮质注射后3天建立MCAO模型,所有组于再灌注后24h,采用Western blot检测脑组织自噬相关蛋白LC3II/I表达水平;免疫荧光双标观察LC3与神经元的共表达情况;透射电镜观察自噬体的形成情况;神经功能缺损评分估计神经功能恢复情况,TTC染色法检测脑梗死体积。然后采用Co-IP检测RGMa干预后Beclin1与PIK3C3结合有无改变;Co-IP检测缺血再灌注后RGMa是否与Beclin1或PIK3C3结合。结果:1.大鼠脑缺血再灌注后24h,I/R组、I/R+NS组及I/R+r Ad-HK组较sham组自噬相关蛋白LC3II/I水平升高(P0.05)、自噬体形成增多、脑梗死体积扩大(P0.05)、神经功能评分降低(P0.05)、;I/R+6FNIII组较I/R组、I/R+NS组自噬相关蛋白LC3II/I水平降低(P0.05)、自噬体形成减少、脑梗死体积缩小(P0.05)、神经功能评分增高(P0.05);I/R+r Ad-sh RGMa组较I/R组、I/R+NS组、I/R+r Ad-HK组自噬相关蛋白LC3II/I水平降低(P0.05)、自噬体形成减少、脑梗死体积缩小(P0.05)、神经功能评分增高(P0.05);而I/R+NS组、I/R+r Ad-HK组较I/R组以上各项均无统计学意义(P0.05)。2.大鼠脑缺血再灌注后24h,I/R组RGMa与Beclin1及PIK3C3均无结合;I/R+6FNIII组可见Beclin1与PIK3C3结合。结论:在缺血再灌注急性期,外源性6FNIII可有效抑制RGMa生物学作用的发挥。RGMa干预后自噬水平降低、促进神经功能恢复,提示脑梗死急性期RGMa可能通过促进自噬而参与抑制神经功能恢复的过程。缺血再灌注后RGMa与Beclin1及PIK3C3均无结合,RGMa干预后仍可见Beclin1与PIK3C3结合,提示脑梗死急性期RGMa可能通过Beclin1非依耐途径参与自噬的调控。
[Abstract]:Background and purpose: cerebrovascular disease is a common disease which is very dangerous to human health. It is one of the three most fatal diseases of human being, and ischemic cerebrovascular disease is the main type. Cerebral ischemia will cause brain tissue damage, and cerebral ischemia reperfusion process often aggravates this damage. Autophagy (autophagy) is a eukaryotic cell. A highly conserved cell that forms vesicles and combines with lysosomes to degrade long acting proteins, damaged proteins and damaged organelles, the metabolism of cells, the development of nutrients and energy, and the process of maintaining intracellular homeostasis. A large number of studies have found that cerebral ischemia reperfusion can induce autophagy, and the process of autophagy mainly occurs in neurons, mainly with hunger. Starvation, lack of growth factor and infection of pathogens are important links of cerebral ischemia reperfusion injury. This process is regulated by a series of complex signal molecules, in which Beclin1 is one of the most important molecules in autophagy regulation pathway. It is combined with PIK3C3 to form Beclin1-PIK3C3 complex, and is widely involved in the formation and maturation of autophagic. Few researchers have found that the target protein affects autophagy by acting on the Beclin1-PIK3C3 complex, known as "Beclin1 dependent autophagy," and some researchers found that the target protein does not affect autophagy through the action of Beclin1. This process is called "Beclin1 independent autophagy." in recent years there has been a study of cerebral ischemia and reperfusion. Beclin1 non dependent autophagy may be associated with the level of apoptosis and the inhibition of the recovery of brain function. Therefore, autophagy may become an important target for the treatment of cerebral ischemic disease in the future. The rejection guide molecule a (Repulsive guidance molecule a, RGMa) is a kind of repellent axon guiding molecule, and a large number of studies have been made. Now, the expression of RGMa increases after cerebral ischemia and reperfusion, which is due to the existence of a series of axon growth inhibitors such as RGMa, which makes the neuron repair and neural function recovery of the central nervous system (CNS) damaged extremely difficult. Our previous study found that the intervention of RGMa expression after cerebral ischemia injury in rats could significantly improve the cerebral ischemia reperfusion injury. The growth of the axon injury and the effective promotion of neural function recovery.6FNIII are 6 fibrin III like domains of the RGMa receptor neogenin molecule. Exogenous 6FNIII can combine with RGMa molecules in vivo to inhibit its biological function. This experiment uses 6FNIII to interfere with RGMa/Neogenin pathway and recombinant adenovirus to inhibit RGMa The effect of RGMa on the autophagy and the recovery of neural function induced by cerebral cortical neuron ischemia reperfusion in rats was investigated by two means of protein expression. At the same time, preliminary exploration was made on the mechanism of RGMa affecting autophagy. Method: 1. 30 adult male adult SD rats were randomly divided into 5 groups: sham operation group (sham), cerebral ischemia reperfusion group (I/R), Cerebral ischemia reperfusion and low concentration 6FNIII intervention group (I/R+L-6FNIII), cerebral ischemia reperfusion and middle concentration 6FNIII intervention group (I/R+M-6FNIII), cerebral ischemia reperfusion and high concentration 6FNIII intervention group (I/R+H-6FNIII), stereotactic lateral ventricle injection of 6FN III of different concentration of 6FN III, immediately established the middle cerebral artery occlusion (MCAO) model, all groups were re After perfusion 24h, Western-blot was used to detect the expression of CRMP-2 protein in the downstream of RGMa, and the best 6FNIII intervention concentration.2. was determined to divide 108 adult male adult SD rats into 6 groups randomly: sham operation group (sham), cerebral ischemia reperfusion group (I/R), cerebral ischemia reperfusion plus physiological saline injection group (I/R+NS), cerebral ischemia-reperfusion plus 6FNIII intervention group (I/R+6FNI) II), cerebral ischemia reperfusion and adenovirus injection group (I/R+r Ad-HK) and cerebral ischemia reperfusion plus RGMa recombinant adenovirus intervention group (I/R+r Ad-sh RGMa).6FNIII intervention group and NS injection group were injected immediately after the 6FNIII or NS lateral ventricle injection, and the adenovirus intervention group established the model 3 days after the injection of the cerebral cortex of the virus. After reperfusion 24h, Western blot was used to detect the expression of autophagy related protein LC3II/I expression in brain tissue; the co expression of LC3 and neurons was observed by double immunofluorescence; transmission electron microscopy was used to observe the formation of autophagic body; neural function defect score was used to estimate the recovery of nerve function, and TTC staining method was used to detect the volume of cerebral infarction. Then Co-I was used to detect the volume of cerebral infarction. Then Co-I was used to detect the volume of cerebral infarction. P detected the combination of Beclin1 and PIK3C3 after RGMa intervention; Co-IP was used to detect the combination of RGMa with Beclin1 or PIK3C3 after ischemia-reperfusion. Results: the levels of autophagic related proteins in the 1. rats were higher than those in the group of 24h, I/R, I/R+NS and I/R+r. The neurological function score was reduced (P0.05), and in the group I/R+6FNIII, the LC3II/I level of autophagic related proteins decreased (P0.05), the formation of autophagic bodies decreased, the volume of cerebral infarction decreased (P0.05) and the neurological function score increased (P0.05) in the group I/R+NS, and the I/R+r Ad-sh RGMa group was lower than that of the I/R group. The volume of body formation decreased, the volume of cerebral infarction was reduced (P0.05), and the score of nerve function increased (P0.05), while in group I/R+NS and I/R+r Ad-HK, there was no statistical significance in group I/R above group I/R (P0.05), and 24h in.2. rats after cerebral ischemia-reperfusion. During the sexual phase, exogenous 6FNIII could effectively inhibit the biological action of RGMa, and the level of autophagy decreased after.RGMa intervention, promoting the recovery of nerve function, suggesting that RGMa may participate in the process of inhibiting the recovery of nerve function by promoting autophagy at acute stage of cerebral infarction. RGMa and Beclin1 and PIK3C3 are not combined after ischemia-reperfusion, and Becli still can be seen after RGMa intervention. The combination of N1 and PIK3C3 suggests that RGMa may participate in the regulation of autophagy through the Beclin1 non dependent pathway in acute phase of cerebral infarction.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743

【参考文献】

相关期刊论文 前10条

1 杜国英;;高同型半胱氨酸致缺血性脑血管病的研究进展[J];医学理论与实践;2017年03期

2 Jiejie Li;Yongjun Wang;;Blood Biomarkers in Minor Stroke and Transient Ischemic Attack[J];Neuroscience Bulletin;2016年05期

3 幸享凤;王恬竹;秦新月;;CRMP2可通过改善神经细胞凋亡减轻缺血/再灌注大鼠神经功能缺损[J];中国药理学通报;2016年04期

4 郭鑫;杨俊;;Beclin1调控自噬、凋亡与炎症反应的分子机制[J];中国老年学杂志;2014年18期

5 王恬竹;秦新月;;排斥导向分子在中枢神经系统中的研究进展[J];西部医学;2014年06期

6 韩侨宇;江昭;陈春花;;自噬在脑缺血损伤中的作用研究[J];解剖科学进展;2013年05期

7 杨吉平;苟兴春;徐曦;王爽;赵朝华;;自噬作用在阿尔茨海默病中的分子机制[J];解剖科学进展;2012年02期

8 乜全民;张世明;;大鼠局灶性脑缺血再灌注损伤后自噬的初步研究[J];中国临床神经科学;2010年01期

9 尹红蕾;秦新月;;RGMa及其在缺血性脑损伤后中枢神经再生中的作用[J];神经损伤与功能重建;2008年03期

10 黄平;刘岩峰;托娅;张平;丁存晶;方杰;王振原;;大鼠弥漫性脑损伤后S100β表达与损伤时间关系[J];法医学杂志;2006年01期

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