CX3CR1在缺血性白质损伤中的意义及机制研究

发布时间：2018-02-11 14:02

本文关键词： 脑低灌注白质 CX3CR1 认知 p38MAPK　出处：《吉林大学》2014年博士论文　论文类型：学位论文

【摘要】：缺血性白质损伤可以引起认知功能障碍，严重损害人类健康，其主要的发生机制有免疫炎性损害、氧化应激损伤、突触结构及功能异常等。目前研究的较多的为免疫炎性损伤机制。小胶质细胞作为中枢神经系统免疫吞噬细胞发挥着双重作用，少量被激活时，可以释放神经营养因子、吞噬废弃的组织碎片发挥保护性作用；大量被激活则产生炎性损害作用。CX3CR1作为小胶质细胞表面的一种受体分子，在生理条件下，使小胶质细胞保持静息状态、维持内环境的稳定状态；在急性缺血、变性疾病、神经创伤时发挥着损害性作用。在缺血性白质损伤时，如何变化、起什么样的作用尚未有研究。p38MAPK是MAPK家族中重要成员，主要参与炎性应激反应。p38MAPK是否参与CX3CR1介导的缺血性白质损伤的发病过程目前还不清楚。目的：观察CX3CR1是否参与了慢性缺血性白质损伤发病过程；CX3CR1对慢性缺血性白质损伤是否通过p38MAPK途经实现。方法：雄性Wistar大鼠320只，随机分为正常组、假手术组、缺血组、阴性对照组、干预组，Morris水迷宫观察造模后28d学习记忆功能。造模后不同时间点（1d、3d、7d、14d、28d），HE染色及Luxol Fast Blue（LFB）染色法评价胼胝体、内囊、视神经纤维变化；Western Blot观察CX3CR1、P-p38/p38蛋白表达；免疫荧光双标技术观察CX3CR1与小胶质细胞关系；免疫组织化学染色评价小胶质细胞变化规律；侧脑室内注射不同浓度CX3CR1中和抗体、p38抑制剂进一步观察缺血后上述指标的变化。结果：（1）造模后28d，缺血组逃避潜伏期、空间探索路径较正常组延长，跨越平台次数显著减少（P0.01）。（2）随着缺血时间的延长，HE染色观察到胼胝体、内囊及视神经区域白质纤维疏松，，排列紊乱且空泡样改变；LFB染色可见上述区域髓鞘崩解范围扩大，分层明显，部分髓鞘出现空泡状；CX3CR1表达量持续增加，与CD11b标记的小胶质细胞数目持续增多相一致且高于假手术组和正常组（P0.01）。（3）随着缺血时间的延长，P-p38/p38比值在缺血3d时出现明显变化，与CX3CR1变化具有相关性。（4）不同浓度CX3CR1中和抗体对缺血性白质损伤的认知功能、纤维损伤程度、CD11b小胶质细胞数目及P-p38/p38比值均有改善作用，且呈现剂量依赖性。应用p38抑制剂后，缺血性白质的纤维损伤程度及小胶质细胞数目均有不同程度改善。结论：（1）CX3CR1过表达通过介导小胶质细胞的变化对缺血性白质产生损伤作用，进而影响空间学习记忆功能。（2）CX3CR1/p38MAPK信号通路是缺血性白质损伤发生机制之一。
[Abstract]:Ischemic white matter injury can cause cognitive impairment and serious damage to human health. Its main mechanisms are immune inflammatory damage and oxidative stress injury. Abnormal synaptic structure and function. The mechanism of immune inflammatory injury is studied. Microglia, as immune phagocytes of the central nervous system, play a dual role, when a small amount of activated, can release neurotrophic factors, Phagocytosis of abandoned tissue fragments plays a protective role; a large number of activated inflammatory damage. CX3CR1, as a receptor molecule on the surface of microglia, keeps the microglia in a resting state under physiological conditions. Maintain a stable state of the internal environment; play a damaging role in acute ischemia, degenerative diseases, and neurotrauma. In ischemic white matter injury, how and what role to play has not been studied. P38 MAPK is an important member of the MAPK family. It is unclear whether p38 MAPK is involved in the pathogenesis of ischemic white matter injury mediated by CX3CR1. Objective: to observe whether CX3CR1 was involved in the pathogenesis of chronic ischemic white matter injury (CX3CR1) and whether it was achieved by p38 MAPK. Methods: 320 male Wistar rats were randomly divided into normal group, sham operation group and ischemic group. In the negative control group, the learning and memory function was observed by Morris water maze on the 28th day after modeling, and the expression of CX3CR1P-p38 / p38 protein was evaluated by Luxol Fast BlueLFB staining and Luxol Fast BlueLFB staining at different time points. The expression of CX3CR1P-p38 / p38 protein was evaluated by Western Blot in corpus callosum, internal capsule and optic nerve fibers. The relationship between CX3CR1 and microglia was observed by immunofluorescence double labeling technique, and the changes of microglia were evaluated by immunohistochemical staining. The changes of above indexes after ischemia were further observed by intraventricular injection of different concentrations of CX3CR1 neutralizing antibody p38 inhibitor. Results the escape latency and spatial exploration pathway of ischemia group were longer than that of normal group 28 days after modeling. With the prolongation of ischemia time, the white matter fibers of corpus callosum, internal capsule and optic nerve region were loose, and the disordered arrangement and vacuolar change LFB staining showed that the area of myelin disintegrating was enlarged. The expression of CX3CR1 was increased in some myelin sheath. The number of microglia labeled with CD11b was increased and higher than that of sham operation group and normal group (P 0.01). The ratio of P-p38 / p38 was significantly changed with the prolongation of ischemic time on the 3rd day after ischemia. The cognitive function of different concentrations of CX3CR1 neutralizing antibody to ischemic white matter injury, the number of CD11b microglia and the ratio of P-p38 / p38 were improved in a dose-dependent manner. The degree of fibrous injury and the number of microglia in ischemic white matter were improved in different degrees. Conclusion the overexpression of CX3CR1 can induce damage to ischemic white matter by mediating the change of microglia. Furthermore, affecting the spatial learning and memory function of CX3CR1 / p38 MAPK signaling pathway is one of the mechanisms of ischemic white matter injury.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R742

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