本文選題：PD-L1 + 腦出血　； 參考：《天津醫(yī)科大學》2017年碩士論文

【摘要】：背景和目的:腦出血(intracerebral hemorrhage,ICH)是臨床上一類致死率高、致殘率高的腦血管病,當前腦出血的發(fā)病率大約為24.6人/10萬人/年,預計到2050年為止發(fā)病率將增加一倍。目前對于腦出血還沒有特異性強的藥物或手術治療方法,臨床上主要以對癥支持治療為主。早期腦損傷主要是由于血腫的占位效應,但病情最嚴重的時期往往在腦出血后的第2-3天,此時炎癥反應達到高峰。越來越多國內外研究的結論顯示,在腦出血后造成神經功能缺損加重的最主要因素是炎癥反應導致的繼發(fā)性腦損傷。腦出血發(fā)生后,位于中樞的小膠質細胞被激活,并釋放促炎介質引起外周白細胞向中樞遷移、浸潤,血腦屏障完整性亦受到破壞。程序性死亡受體1(programmed death 1,PD-1)是一類跨膜糖蛋白,屬于CD28/CTLA-4免疫球蛋白超家族。它的配體有兩種,分別為程序性死亡配體1(Programmed Death Ligand 1,PD-L1)與程序性死亡配體2(Programmed Death Ligand 2,PD-L2)。為了了解PD-1/PD-L1在腦出血后炎癥反應中的作用,本研究以ICH小鼠為對象,觀察PD-L1及其抗體在腦出血損傷中的作用并初步探討相關機制。方法:自體血30μl注入雄性C57BL/6 J小鼠建立腦出血模型。小鼠隨機分配到對照組、PD-L1蛋白組和PD-L1抗體組。腦出血后一小時,分別對三組小鼠腹腔注射磷酸鹽緩沖液(PBS)、PD-L1蛋白和PD-L1抗體。之后進行神經功能學評價,在腦出血后的第三天,即炎癥反應達到高峰時,稱量腦組織干濕重來評估腦水腫程度;對小鼠腦組織取材,通過流式細胞分析術研究腦組織中各類免疫細胞的浸潤情況;通過實時-聚合酶鏈式反應(RT-PCR)測定腦內及外周炎癥因子水平;提取腦組織總蛋白,進行蛋白印跡分析,測定腦組織緊密連接蛋白水平來評估血腦屏障完整性,并探討PD-L1的作用通路。結果:PD-L1顯著改善了腦出血后的神經功能缺損癥狀,減輕了腦水腫程度并且減小了血腫體積。PD-L1降低了腦出血后腦組織中浸潤的CD4+T細胞數(shù)量,在CD4+T細胞亞群中降低了Th1和Th17細胞的比例,使Th2和調節(jié)性T細胞(Treg)的比例增加。在PD-L1蛋白組中,亦可觀察到炎癥環(huán)境的改善,以及血腦屏障完整性的增強。PD-L1也抑制mTOR通路。在ICH小鼠中使用PD-L1抗體后顯示與PD-L1蛋白相反的作用。結論:PD-L1主要是通過抑制mTOR通路來調節(jié)CD4+T細胞數(shù)量,影響Treg/Th17細胞平衡,從而減輕腦出血后的腦水腫程度,并改善腦出血后神經功能。相反地,PD-L1抗體加重腦出血周圍腦組織的炎癥反應,導致ICH小鼠病情的惡化。
[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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R743.34

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