缺血性脑损伤中TLR2介导的MyD88信号调控机制的研究
发布时间：2018-10-29 09:15
【摘要】：在许多国家,卒中已成为了主要的致死原因。在中国县乡,脑血管病为第二位致死原因;在中国的大城市,脑血管病是第三位致死原因。脑卒中后的残疾及功能障碍给患者与家庭带来了极大的负担。卒中患者中大部分为缺血性卒中,少部分为出血性卒中。分布于免疫细胞、血管内皮细胞以及脑组织固有细胞等表面的跨膜TLRs可能介导脑缺血后的炎症反应。研究显示,TLR2在脑缺血后数小时即上调,可能参与了脑缺血后神经元损伤。Ziegler等研究表明,MCAO后注射抗TLR2封闭性单克隆抗体T2.5,免疫组化发现CD1 1b阳性细胞数量下降,还能抑制白细胞聚集以及小胶质细胞迁移;同时导致脑缺血后NeuN阳性细胞数量增加,提示抑制TLR2具有神经保护作用。髓样分化因子(MyD88)是TLR信号通路中的重要转导蛋白,其依赖的信号通路以及调控的基因产物在固有免疫和适应性免疫中发挥着关键作用。基质金属蛋白酶9(MMP-9)可降解脑血管周围基膜的成分从而导致BBB通透性增加,白细胞侵润、脑水肿及出血转化。目的:探讨大鼠脑缺血再灌注后,Toll样受体2(Toll-like receptor 2,TLR2)、髓样分化因子88(Myeloid differentiation factor 88,MyD88)与基质金属蛋白酶9(matrix metalloproteinase-9,MMP-9)三者之间的变化关系,以及三者在脑缺血再灌注后脑损伤形成过程中的可能作用机制。方法:制作Sprague-Dawley大鼠(250-280g)右侧大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)2小时再灌注模型。实验共分为三组:缺血组、sham组(对照组)、T2.5处理组(大鼠MCAO 2h再灌注开始时,经颈静脉注射T2.5,剂量为0.1212μg/g)。在脑缺血再灌注后不同时间点,选取缺血组、sham组和T2.5处理组缺血侧脑组织来进行以下实验:1、MCAO 再灌注 1h、2h、3h、6h、12h、24h 六个时间点,利用 western blot来测定sham组和缺血组缺血侧大脑皮层中TLR2、MyD88及MMP-9的蛋白表达水平变化情况(每个时间点五只老鼠,n=5)。2、MCAO再灌注开始时,给予TLR2拮抗剂T2.5处置。缺血再灌注24h时,利用western blot测定缺血组和T2.5处理组缺血侧大脑皮层中TLR2、MyD88及MMP-9的蛋白表达水平变化情况(该时间点五只老鼠,n=5)。3、MCAO再灌注24h后,测定三组中的脑梗死体积(TTC染色法)、脑水肿(干湿重法)、血脑屏障(blood-brain barrier,BBB)通透性(Evan's蓝法)以及神经功能缺损评分(每种测量五只老鼠,n=5)。结果:1、Western blot结果显示,在大鼠MCAO再灌注6h,缺血组缺血侧皮层中TLR2较sham组即开始升高,且有显著性差异(p0.05),持续至24h(p0.05)。2、Western blot结果显示,在大鼠MCAO再灌注6h,缺血组缺血侧皮层中MyD88较sham组即开始升高,且有显著性差异(p0.01),持续至24h(p0.05)。3、Western blot结果显示,在大鼠MCAO再灌注24h,缺血组缺血侧皮层中MMP-9较sham组升高,具有显著性差异(p0.05)。4、大鼠MCAO再灌注24h,缺血组缺血侧脑组织BBB通透性,脑水肿程度,脑梗死体积及神经功能缺损评分均较sham组升高,且有显著性差异(p0.01、p0.01、p0.001、p0.001)。5、Western blot结果显示,大鼠MCAO再灌注24h,T2.5处理组与缺血组比较,TLR2、MyD88及MMP-9表达水平均降低(p0.05)。大鼠MCAO再灌注24h,T2.5处理组BBB通透性、脑水肿程度、脑梗死体积、神经功能缺损评分均较缺血组下降,且均有显著性差异(p0.01、p0.05、p0.01、p0.01)。结论:1、脑缺血再灌注后6h,TLR2及MyD88即开始上升;脑缺血再灌注后24h,MMP-9才开始上升,以上因素可能通过增加BBB通透性,参与了脑水肿、脑梗死及神经功能损伤。2、TLR2拮抗剂T2.5可能通过拮抗TLR2-MyD88信号通路,减少MMP-9的表达,从而减轻BBB通透性,缓解脑水肿、减少脑梗死及修复神经功能,最终改善缺血再灌注脑损伤。
[Abstract]:Stroke has become a major cause of death in many countries. In China, cerebrovascular disease is the second cause of death; in big cities in China, cerebrovascular disease is the third cause of death. Disability and dysfunction after stroke have a great burden on the patient and the family. Most of stroke patients are ischemic stroke and a small part is hemorrhagic stroke. Transmembrane TLRs distributed on the surface of immune cells, vascular endothelial cells and native cells of brain tissue may mediate inflammatory responses following cerebral ischemia. The study showed that TLR2 was upregulated after focal cerebral ischemia and may be involved in neuronal damage after cerebral ischemia. Ziegler et al. showed that the number of CD1b positive cells decreased and the number of CD1b positive cells decreased, and the number of N positive cells increased after cerebral ischemia. It is suggested that inhibition of TLR2 has neuroprotective effect. The myeloid differentiation factor (MyD88) is an important transduction protein in TLR signaling pathway, and its dependent signaling pathway and regulated gene products play a key role in innate immunity and adaptive immunity. Matrix metalloproteinase-9 (MMP-9) can degrade the components of basement membrane around cerebral vessels, which leads to increased permeability of the brain, invasion of leucocytes, cerebral edema and hemorrhagic transformation. Objective: To investigate the changes of Toll-like receptor 2 (TLR2), myeloid differentiation factor (88, MyD88) and matrix metalloproteinase-9 (MMP-9) after cerebral ischemia-reperfusion in rats. Methods: A 2-hour reperfusion model was established in the right cerebral artery of Sprague-Dawley rats (250-280g). The experiment was divided into three groups: ischemic group, sham group (control group), and T2. 5 treatment group (at the beginning of reperfusion in the rat heart O 2h, the dose was 0. 1212 ug/ g). At different time points after cerebral ischemia-reperfusion, ischemia group, sham group and T2. 5 treatment group were selected for the following experiments: 1, 1h, 2h, 3h, 6h, 12h and 24h were reperfusion 1h, 2h, 3h, 6h, 12h and 24h, and the TLR2 in cerebral cortex of ischemic side was determined by western blot. The levels of protein expression in MyD88 and MMP-9 (five rats per time point, n = 5). 2. At the start of reperfusion, TLR2 antagonist T2. 5 was administered. The levels of TLR2, MyD88 and MMP-9 in cerebral cortex of ischemic-side cerebral cortex were determined by western blot. The levels of TLR2, MyD88 and MMP-9 in ischemic-side cerebral cortex were determined by western blot. Brain edema (dry-wet weight method), blood-brain barrier (BBB) permeability (Evan's blue method) and neurological deficit score (five mice each, n = 5). Results: 1. Western blot analysis showed that there was a significant difference (P0.05) in the ischemia-side cortex of rats after reperfusion 6h, and there was significant difference (P0.05). There was a significant difference in the expression of MMP-9 in the ischemic side cortex of the ischemic group (P0.05). The results of Western blot showed that the MMP-9 was higher in the ischemic side cortex of the ischemia group than in the sham group (P0.05). The permeability of brain tissue, the degree of cerebral edema, the volume of cerebral infarction and neurological deficit were higher in ischemic group than sham group, and there was a significant difference (P. 01, No. 01, No. 001, V1.001). The results of Western blot showed that the reperfusion 24h, T2. 5 treatment group in rats were compared with ischemia group and TLR2. The expression levels of MyD88 and MMP-9 decreased (P0.05). The permeability, degree of brain edema, volume of cerebral infarction and neurological deficit were all lower than that in ischemia group. Conclusion: 1. After cerebral ischemia and reperfusion, 6h, TLR2 and MyD88 started to rise; after cerebral ischemia and reperfusion 24h, MMP-9 began to rise. The above factors may be involved in cerebral edema, cerebral infarction and nerve function injury by increasing permeability of TLR2. The TLR2 antagonist, T2. 5, may antagonize the signal pathway of TLR2-MyD88. the expression of MMP-9 is reduced, so that the permeability of the brain is reduced, the cerebral edema is relieved, the cerebral infarction is reduced, the nerve function is restored, and the brain injury of the ischemia reperfusion is finally improved.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743
本文编号：2297272
[Abstract]:Stroke has become a major cause of death in many countries. In China, cerebrovascular disease is the second cause of death; in big cities in China, cerebrovascular disease is the third cause of death. Disability and dysfunction after stroke have a great burden on the patient and the family. Most of stroke patients are ischemic stroke and a small part is hemorrhagic stroke. Transmembrane TLRs distributed on the surface of immune cells, vascular endothelial cells and native cells of brain tissue may mediate inflammatory responses following cerebral ischemia. The study showed that TLR2 was upregulated after focal cerebral ischemia and may be involved in neuronal damage after cerebral ischemia. Ziegler et al. showed that the number of CD1b positive cells decreased and the number of CD1b positive cells decreased, and the number of N positive cells increased after cerebral ischemia. It is suggested that inhibition of TLR2 has neuroprotective effect. The myeloid differentiation factor (MyD88) is an important transduction protein in TLR signaling pathway, and its dependent signaling pathway and regulated gene products play a key role in innate immunity and adaptive immunity. Matrix metalloproteinase-9 (MMP-9) can degrade the components of basement membrane around cerebral vessels, which leads to increased permeability of the brain, invasion of leucocytes, cerebral edema and hemorrhagic transformation. Objective: To investigate the changes of Toll-like receptor 2 (TLR2), myeloid differentiation factor (88, MyD88) and matrix metalloproteinase-9 (MMP-9) after cerebral ischemia-reperfusion in rats. Methods: A 2-hour reperfusion model was established in the right cerebral artery of Sprague-Dawley rats (250-280g). The experiment was divided into three groups: ischemic group, sham group (control group), and T2. 5 treatment group (at the beginning of reperfusion in the rat heart O 2h, the dose was 0. 1212 ug/ g). At different time points after cerebral ischemia-reperfusion, ischemia group, sham group and T2. 5 treatment group were selected for the following experiments: 1, 1h, 2h, 3h, 6h, 12h and 24h were reperfusion 1h, 2h, 3h, 6h, 12h and 24h, and the TLR2 in cerebral cortex of ischemic side was determined by western blot. The levels of protein expression in MyD88 and MMP-9 (five rats per time point, n = 5). 2. At the start of reperfusion, TLR2 antagonist T2. 5 was administered. The levels of TLR2, MyD88 and MMP-9 in cerebral cortex of ischemic-side cerebral cortex were determined by western blot. The levels of TLR2, MyD88 and MMP-9 in ischemic-side cerebral cortex were determined by western blot. Brain edema (dry-wet weight method), blood-brain barrier (BBB) permeability (Evan's blue method) and neurological deficit score (five mice each, n = 5). Results: 1. Western blot analysis showed that there was a significant difference (P0.05) in the ischemia-side cortex of rats after reperfusion 6h, and there was significant difference (P0.05). There was a significant difference in the expression of MMP-9 in the ischemic side cortex of the ischemic group (P0.05). The results of Western blot showed that the MMP-9 was higher in the ischemic side cortex of the ischemia group than in the sham group (P0.05). The permeability of brain tissue, the degree of cerebral edema, the volume of cerebral infarction and neurological deficit were higher in ischemic group than sham group, and there was a significant difference (P. 01, No. 01, No. 001, V1.001). The results of Western blot showed that the reperfusion 24h, T2. 5 treatment group in rats were compared with ischemia group and TLR2. The expression levels of MyD88 and MMP-9 decreased (P0.05). The permeability, degree of brain edema, volume of cerebral infarction and neurological deficit were all lower than that in ischemia group. Conclusion: 1. After cerebral ischemia and reperfusion, 6h, TLR2 and MyD88 started to rise; after cerebral ischemia and reperfusion 24h, MMP-9 began to rise. The above factors may be involved in cerebral edema, cerebral infarction and nerve function injury by increasing permeability of TLR2. The TLR2 antagonist, T2. 5, may antagonize the signal pathway of TLR2-MyD88. the expression of MMP-9 is reduced, so that the permeability of the brain is reduced, the cerebral edema is relieved, the cerebral infarction is reduced, the nerve function is restored, and the brain injury of the ischemia reperfusion is finally improved.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743
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