程序性死亡配体1对脑出血损伤的作用及机制研究

发布时间：2018-04-10 20:38

本文选题：PD-L1 + 脑出血　；参考：《天津医科大学》2017年硕士论文

【摘要】：背景和目的:脑出血(intracerebral hemorrhage,ICH)是临床上一类致死率高、致残率高的脑血管病,当前脑出血的发病率大约为24.6人/10万人/年,预计到2050年为止发病率将增加一倍。目前对于脑出血还没有特异性强的药物或手术治疗方法,临床上主要以对症支持治疗为主。早期脑损伤主要是由于血肿的占位效应,但病情最严重的时期往往在脑出血后的第2-3天,此时炎症反应达到高峰。越来越多国内外研究的结论显示,在脑出血后造成神经功能缺损加重的最主要因素是炎症反应导致的继发性脑损伤。脑出血发生后,位于中枢的小胶质细胞被激活,并释放促炎介质引起外周白细胞向中枢迁移、浸润,血脑屏障完整性亦受到破坏。程序性死亡受体1(programmed death 1,PD-1)是一类跨膜糖蛋白,属于CD28/CTLA-4免疫球蛋白超家族。它的配体有两种,分别为程序性死亡配体1(Programmed Death Ligand 1,PD-L1)与程序性死亡配体2(Programmed Death Ligand 2,PD-L2)。为了了解PD-1/PD-L1在脑出血后炎症反应中的作用,本研究以ICH小鼠为对象,观察PD-L1及其抗体在脑出血损伤中的作用并初步探讨相关机制。方法:自体血30μl注入雄性C57BL/6 J小鼠建立脑出血模型。小鼠随机分配到对照组、PD-L1蛋白组和PD-L1抗体组。脑出血后一小时,分别对三组小鼠腹腔注射磷酸盐缓冲液(PBS)、PD-L1蛋白和PD-L1抗体。之后进行神经功能学评价,在脑出血后的第三天,即炎症反应达到高峰时,称量脑组织干湿重来评估脑水肿程度;对小鼠脑组织取材,通过流式细胞分析术研究脑组织中各类免疫细胞的浸润情况;通过实时-聚合酶链式反应(RT-PCR)测定脑内及外周炎症因子水平;提取脑组织总蛋白,进行蛋白印迹分析,测定脑组织紧密连接蛋白水平来评估血脑屏障完整性,并探讨PD-L1的作用通路。结果:PD-L1显著改善了脑出血后的神经功能缺损症状,减轻了脑水肿程度并且减小了血肿体积。PD-L1降低了脑出血后脑组织中浸润的CD4+T细胞数量,在CD4+T细胞亚群中降低了Th1和Th17细胞的比例,使Th2和调节性T细胞(Treg)的比例增加。在PD-L1蛋白组中,亦可观察到炎症环境的改善,以及血脑屏障完整性的增强。PD-L1也抑制mTOR通路。在ICH小鼠中使用PD-L1抗体后显示与PD-L1蛋白相反的作用。结论:PD-L1主要是通过抑制mTOR通路来调节CD4+T细胞数量,影响Treg/Th17细胞平衡,从而减轻脑出血后的脑水肿程度,并改善脑出血后神经功能。相反地,PD-L1抗体加重脑出血周围脑组织的炎症反应,导致ICH小鼠病情的恶化。
[Abstract]:Background and objective: intracerebral hemorrhage (ICH) is a kind of cerebrovascular disease with high mortality and high disability rate. The current incidence of intracerebral hemorrhage is about 24.6 people / 100000 people per year. It is estimated that the incidence rate will double by 2050.At present, there is no specific drug or surgical treatment for intracerebral hemorrhage.Early brain injury is mainly due to the space-occupying effect of hematoma, but the most serious stage is usually at 2-3 days after intracerebral hemorrhage, and the inflammatory reaction reaches the peak at this time.More and more studies at home and abroad show that secondary brain injury caused by inflammatory reaction is the most important factor that results in the aggravation of nerve function defect after intracerebral hemorrhage.After intracerebral hemorrhage the microglia located in the center were activated and inflammatory mediators were released which led to the migration and infiltration of peripheral white blood cells to the center and the damage to the integrity of the blood-brain barrier.1(programmed death 1 PD-1) is a transmembrane glycoprotein, belonging to the CD28/CTLA-4 immunoglobulin superfamily.There are two kinds of ligands, 1(Programmed Death Ligand 1PD-L1 and 2(Programmed Death Ligand 2PD-L2, respectively.In order to understand the role of PD-1/PD-L1 in the inflammatory response after intracerebral hemorrhage (ICH), the role of PD-L1 and its antibodies in ICH injury was observed in ICH mice and the related mechanisms were preliminarily investigated.Methods: intracerebral hemorrhage model was established by injecting 30 渭 l autologous blood into male C57BL/6 J mice.Mice were randomly assigned to control group PD-L1 protein group and PD-L1 antibody group.One hour after intracerebral hemorrhage, three groups of mice were intraperitoneally injected with phosphate buffer solution PBS-1 protein and PD-L1 antibody.Then the neurological function was evaluated. On the third day after intracerebral hemorrhage, when the inflammatory reaction reached its peak, the brain tissue was weighed with dry and wet weight to assess the degree of brain edema.Flow cytometry was used to study the infiltration of various kinds of immune cells in brain tissue, real-time polymerase chain reaction (RT-PCR) was used to determine the level of inflammatory factors in the brain and peripheral inflammatory factors were detected, and the total protein of brain tissue was extracted and analyzed by Western blotting.To evaluate the integrity of blood-brain barrier and to explore the pathway of PD-L1, the level of tight junction protein in brain tissue was measured to evaluate the integrity of blood-brain barrier.Results: after intracerebral hemorrhage, the symptom of neurological deficit was significantly improved, the degree of cerebral edema was alleviated, and the volume of hematoma. PD-L1 decreased the number of infiltrated CD4 T cells in brain tissue after ICH.The proportion of Th1 and Th17 cells was decreased in CD4 T cell subsets, and the proportion of Th2 and regulatory T cells was increased.In PD-L1 protein group, the improvement of inflammatory environment and the enhancement of blood-brain barrier integrity. PD-L1 also inhibited the mTOR pathway.The use of PD-L1 antibody in ICH mice showed the opposite effect of PD-L1 protein.Conclusion it is suggested that the regulation of the number of CD4 T cells and the balance of Treg/Th17 cells through the inhibition of mTOR pathway may reduce the degree of cerebral edema and improve the neurological function after intracerebral hemorrhage.On the contrary, PD-L1 antibody aggravated the inflammatory reaction of brain tissue around cerebral hemorrhage, leading to the deterioration of ICH mice.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.34

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