早期运动训练对脑缺血损伤大鼠的脑保护作用及其机制研究

发布时间：2018-03-01 10:14

本文关键词： 早期运动训练脑缺血损伤血脑屏障神经元凋亡脑保护　出处：《复旦大学》2014年博士论文　论文类型：学位论文

【摘要】：研究背景：缺血性脑卒中是具有严重破坏性的急性脑血管疾病,它可以激活脑缺血瀑布,破坏血脑屏障,引发免疫炎症反应以及缺血区域神经元凋亡,从而导致脑缺血继发性神经功能损伤。大量的基础和临床研究证实早期运动训练促进卒中患者的神经功能恢复,但其所涉及的分子机制尚未完全阐明。本研究着眼于脑缺血继发性损伤的两个重要病理过程——血脑屏障破坏和神经元凋亡,拟利用大鼠大脑中动脉栓塞的脑缺血模型(MCAO),在缺血早期介入强度逐步增加的运动康复训练,综合运用行为学、组织学、形态学、分子生物学和蛋白质组学方法研究早期运动训练对脑缺血损伤大鼠血脑屏障完整性和神经元凋亡的影响,探讨早期运动训练的脑保护作用及其潜在的分子机制。研究方法：采用SD成年雄性大鼠,随机分为假手术组、缺血对照组(non-exercise)和早期运动组(early-exercise),建立90分钟缺血时程的MCAO模型,在脑缺血再灌注24小时进行强度渐增的跑台运动训练干预,每天30分钟。在脑缺血损伤后相应时间点,采用神经功能缺失评分法评估大鼠神经功能缺失症状,分别使用foot-fault和beam balance方法评估大鼠的运动协调能力和平衡能力。此外,通过TTC染色和CV染色法检测大鼠脑梗死体积；利用透射电镜技术和伊文思蓝染色研究血脑屏障结构和功能的变化；同时采用Real-Time PCR、western blot和明胶酶谱法分析MMPs、TIMP-1、occludin、TLR4、NF-κB、IL-18的表达变化,研究早期运动对血脑屏障的保护机制；最后,我们采用TUNEL和F-J-B染色观察大鼠损伤侧皮层的神经元凋亡情况；western blot检测神经元凋亡相关蛋白caspase-3和bcl-2的表达情况,进一步探索早期运动的脑保护作用机制。研究结果：早期运动训练显著减轻了脑缺血损伤引起的神经功能缺失。与non-exercise相比,early-exercise组大鼠神经功能缺失症状得到显著缓解,运动协调能力也明显恢复,脑梗死体积减小。电镜结果和伊文思蓝染色显示早期运动减轻脑缺血损伤对血脑屏障的破坏。早期运动抑制了脑缺血损伤引起的MMPs蛋白表达及其活性,促进TIMP-1表达,减少紧密连接蛋白occludin缺失,抑制TLR4、NF-κB、IL-18炎性相关介质的表达,从而发挥了对血脑屏障的保护作用。另外,早期运动通过促进抗凋亡蛋白bcl-2的表达下调caspase-3的核裂解作用,并且运用TUNEL和F-J-B染色也观察到早期运动大鼠损伤侧皮层的神经元凋亡减少,进一步发挥脑保护作用。结论：脑卒中早期运动训练具有脑保护作用,其作用机制可能与抑制TLR4/NF-κB信号传导通路,下调促炎性因子,调控MMPs及其组织抑制因子TIMPs表达,减少occludin缺失,减轻血脑屏障损伤有关；也与早期运动调控凋亡相关蛋白caspase-3和bcl-2,抑制神经元凋亡密切相关。早期运动脑保护作用完整的分子机制仍需要进一步的深入研究,为缺血性脑卒中的临床治疗提供新的思路和理论基础。
[Abstract]:Background: ischemic stroke is a severe and destructive acute cerebrovascular disease. It can activate cerebral ischemic waterfall, destroy blood-brain barrier, induce immune inflammation and neuronal apoptosis in ischemic region. A large number of basic and clinical studies have confirmed that early exercise training promotes the recovery of neurological function in stroke patients. However, the molecular mechanism involved has not been fully elucidated. This study focuses on two important pathological processes of secondary injury of cerebral ischemia-blood-brain barrier damage and neuronal apoptosis. The model of cerebral ischemia in rats with middle cerebral artery embolism (MCAO) was used to train the exercise rehabilitation of increasing interventional intensity in the early stage of ischemia, and to use behavior, histology and morphology. Molecular biology and proteomics methods were used to study the effects of early exercise training on the integrity of blood-brain barrier and neuronal apoptosis in rats with cerebral ischemia injury. Methods: Sprague-Dawley adult male rats were randomly divided into sham-operated group, non-exercise control group and early exercise group to establish a 90-minute ischemic MCAO model. After 24 hours of cerebral ischemia-reperfusion, the rats were treated with increased treadmill exercise training for 30 minutes a day. At the corresponding time points after cerebral ischemia injury, the neurological deficit score was used to evaluate the neurological deficit symptoms in rats. Foot-fault and beam balance were used to evaluate the ability of motor coordination and balance in rats. In addition, the volume of cerebral infarction was detected by TTC staining and CV staining. The changes of blood-brain barrier structure and function were studied by means of transmission electron microscopy and Evans blue staining, and the expression of IL-18 in TLR4NF- 魏 B was analyzed by Real-Time PCR western blot and gelatin zymography to study the protective mechanism of early exercise on blood-brain barrier. TUNEL and F-J-B staining were used to observe the neuronal apoptosis in the injured cortex of rats. The expression of apoptosis-related protein caspase-3 and bcl-2 was detected by western blot. The results showed that early exercise training significantly alleviated the neurological deficit caused by cerebral ischemia injury. Compared with non-exercise, the neurological deficit symptoms of rats in the early exercise group were significantly alleviated. The ability to coordinate sports has also been significantly restored. The results of electron microscope and Evans blue staining showed that early exercise alleviated the damage of blood-brain barrier caused by cerebral ischemia. Early exercise inhibited the expression and activity of MMPs protein and promoted the expression of TIMP-1. By reducing the deletion of tight junction protein occludin and inhibiting the expression of IL-18 inflammatory mediators in TLR4, NF- 魏 B, IL-18 may play a protective role on the blood-brain barrier. In addition, early exercise down-regulates the nuclear cleavage of caspase-3 by promoting the expression of anti-apoptotic protein bcl-2. TUNEL and F-J-B staining were also used to observe the decrease of neuronal apoptosis in the injured lateral cortex of early exercise rats, and to further exert the protective effect of brain. Conclusion: early exercise training has protective effect on brain after stroke. The mechanism may be related to the inhibition of TLR 4 / NF- 魏 B signal transduction pathway, the down-regulation of pro-inflammatory factors, the regulation of MMPs and its tissue inhibitor TIMPs expression, the reduction of occludin deletion and the reduction of blood-brain barrier damage. It is also closely related to the regulation of apoptosis-related proteins caspase-3 and bcl-2 and the inhibition of neuronal apoptosis. The complete molecular mechanism of early motor brain protection still needs to be further studied. To provide a new thinking and theoretical basis for the clinical treatment of ischemic stroke.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743.3

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