手术创伤后外周循环炎症因子对不同年龄小鼠血脑屏障通透性的影响
发布时间：2018-08-04 19:22
【摘要】：血脑屏障(Blood Brain Barrier, BBB)是存在于脑组织与血液之间复杂的细胞系统,能阻挡病原体和有害物质由血液进入脑室和脑组织,是机体天然免疫的重要屏障之一。BBB主要由脑部的微血管内皮细胞(Brain Microvascular Endothelial Cells,BMEC)、周皮细胞(Pericyte)、基膜(Basement Membrance, BM)、星形胶质细胞(Astrocyte)和部分神经元轴突末梢构成。任何组成成分的破坏都将导致BBB通透性的变化,继而影响中枢神经系统(Central Nerves System, CNS)的健康和功能。因此BBB已成为当今医学研究领域热点之一,特别是BBB通透性调控方面的研究对于人类深入认识脑类疾病的致病机制及其治疗具有重大意义。目前国内外对病理因素导致BBB通透性变化的研究都集中于大脑内部的损伤或创伤,如创伤性脑损伤(Traumatic Brain Injury, TBI),中风(Stroke),缺血(Ischemia)等。而外周创伤,如手术,烧创伤等对BBB通透性影响的原因和机制并不明确。以往研究多认为,外周创伤后的应激反应造成中枢神经系统的炎症,进而破坏BBB的完整性.Mervyn等2010年发表在PNAS上的研究证明术前给予小鼠外周循环注射TNF-a中和抗体可以保护BBB的完整性.我们的前期研究也发现,不同年龄组小鼠(9个月和18个月)在经历外周手术(腹部手术)后,血脑屏障通透性随年龄的增长有增加的趋势,且行为学发生了改变,老年组(18个月)的变化更为明显。以上研究均表明,外周循环炎症对手术创伤后BBB通透性存在着影响。因此,寻找出外周创伤导致BBB通透性增高的机制是预防临床术后认知障碍(Postoperate Cognitive Dysfunction, POCD)的关键。基于此,本研究分为以下三部分：1.对本研究采用的小鼠手术模型中可造成血脑屏障通透性增高的因素(麻醉和外周循环中的炎症因子)以及适当检测方法(BBB通透性变化的时间点和示踪剂的选择)进行筛选；2.用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测由手术引起的外周循环中和大脑实质组织炎症因子(IL-6)的浓度,使用分子量为10KDa的示踪剂(10 KDa Tracer)Dextran10,用免疫荧光的方法在荧光显微镜下观察不同年龄组BBB通透性的变化,并对BBB漏出程度予以定量,使用Western Blotting方法检测BBB完整性关键蛋白(紧密连接,Tight Junction,TJ)的变化,使用Y迷宫(YMaze)等行为学检测方法观察小鼠行为学的改变；3.使用白介素-6中和抗体(Anti-IL-6 Neutralizing Antibody)以及使用白介素-6基因敲除小鼠(IL-6 Gene Knockout mice)来验证关键炎症因子导致BBB通透性增高的理论。实验结果显示：1.本研究小鼠在全麻状态下行腹部手术的模型中,外周炎症因子IL-6在手术后6小时左右达到高峰,且老年组(18个月)比普通成年组(9个月)上升幅度更大,而仅给予麻醉组小鼠在24小时内血清IL-6水平以及BBB的通透性没有明显变化；在示踪剂的选择上,仅观察到分子量较小的10KDa的Dextran于在术后6小时左右由脑血管漏出,而分子量较大的70KDa Dextran和伊文氏蓝(Evans Blue,EB)没有漏出；2.对Dextran 10的定量实验中,成年手术组漏出量约为对照组的1.3倍,而老年手术组为1.6倍左右;行为学检测发现术后成年组和老年组小鼠均出现空间记忆力的下降,老年组更为明显；3.术前使用IL-6中和抗体以及IL-6基因敲除鼠的BBB通透性没有变化。以上结果为我们认识机体创伤后,外周循环炎症因子导致BBB通透性增加提供了理论依据和实验基础,也为临床上防治由外周创伤引起的中枢神经系统并发症提供了潜在的治疗方法。
[Abstract]:Blood Brain Barrier (BBB) is a complex cell system that exists between brain tissue and blood. It can block pathogens and harmful substances from the blood into the ventricles and brain tissues. It is one of the important barriers to the natural immune system of the body,.BBB mainly by the microvascular endothelial cells of the brain (Brain Microvascular Endothelial Cells, BMEC), and the pericrp. Pericyte, Basement Membrance, BM, astrocytes (Astrocyte) and part of the axon end of neurons. Destruction of any component will lead to changes in BBB permeability and affect the health and function of the central nervous system (Central Nerves System, CNS). Therefore BBB has become a hot topic in the field of medical research. One point, especially the study of the regulation of BBB permeability, is of great significance for human understanding of the pathogenesis and treatment of brain diseases. At present, the study of pathological factors leading to changes in BBB permeability is concentrated on the injury or trauma in the brain, such as traumatic brain injury (Traumatic Brain Injury, TBI), and stroke (S Troke), ischemia (Ischemia) and so on. But the causes and mechanisms of the effects of peripheral trauma, such as surgery, and trauma, on BBB permeability are not clear. TNF-a neutralization antibody in peripheral circulation can protect the integrity of BBB. Our previous study also found that the blood brain barrier permeability increased with age (9 months and 18 months) after peripheral surgery (abdominal surgery), and the behavioral changes were changed, and the changes in the elderly group (18 months) were more obvious. These studies have shown that peripheral circulation inflammation has an impact on BBB permeability after surgical trauma. Therefore, the key to the prevention of postoperative cognitive impairment (Postoperate Cognitive Dysfunction, POCD) is the key to the prevention of postoperative cognitive impairment (Postoperate Cognitive Dysfunction, POCD). Based on this, the study is divided into the following three parts: 1. In the mouse operation model, the factors that can cause the increase of blood brain barrier permeability (anaesthesia and the inflammatory factors in the peripheral circulation) and the appropriate detection methods (the time point of BBB permeability change and the selection of tracer) are screened; 2. the peripheral blood test (enzyme linked immunosorbent assay, ELISA) is used to detect the circumference caused by the operation. The concentration of inflammatory factors (IL-6) in the circulation and the brain parenchyma (IL-6), using the molecular weight of 10KDa tracer (10 KDa Tracer) Dextran10, was used to observe the changes of BBB permeability in different age groups by fluorescence microscopy, and the degree of BBB leakage was quantified. Western Blotting method was used to detect the key protein of BBB integrity. The changes in the close connection, Tight Junction, TJ), using the Y maze (YMaze) and other behavioral tests to observe the behavioral changes in mice; 3. the use of the interleukin -6 neutralization antibody (Anti-IL-6 Neutralizing Antibody) and the use of the interleukins -6 gene knockout mice (IL-6 Gene) to verify the increase in the permeability of the key inflammatory factors The experimental results showed that 1. in the model of abdominal surgery under general anesthesia, the peripheral inflammatory factor IL-6 reached the peak at about 6 hours after the operation, and the elderly group (18 months) increased more than the normal adult group (9 months), while the serum level of IL-6 and the permeability of BBB in the anesthetic group were only given in the anesthetic group for 24 hours. There was no obvious change in sex; in the selection of tracers, only the Dextran of 10KDa, which was smaller in molecular weight, leaked out of the cerebral vessels about 6 hours after the operation, while the larger molecular weight 70KDa Dextran and Evans blue (Evans Blue, EB) did not leak out; 2. to the quantitative test of Dextran 10, the leakage of the adult operation group was about 1.3 times that of the control group. In the elderly operation group, the operation group was about 1.6 times, and the behavioral test found that there was a decrease in spatial memory in both the adult and the elderly group, and the older group was more obvious. 3. the BBB permeability of the IL-6 neutralization antibody and the IL-6 gene knockout mice before operation was not changed. The results showed that we knew the peripheral circulation inflammatory factors after the body was traumatized. It provides a theoretical basis and experimental basis for the increase of BBB permeability, and also provides a potential treatment for the prevention and treatment of complications of the central nervous system caused by peripheral trauma.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R641
[Abstract]:Blood Brain Barrier (BBB) is a complex cell system that exists between brain tissue and blood. It can block pathogens and harmful substances from the blood into the ventricles and brain tissues. It is one of the important barriers to the natural immune system of the body,.BBB mainly by the microvascular endothelial cells of the brain (Brain Microvascular Endothelial Cells, BMEC), and the pericrp. Pericyte, Basement Membrance, BM, astrocytes (Astrocyte) and part of the axon end of neurons. Destruction of any component will lead to changes in BBB permeability and affect the health and function of the central nervous system (Central Nerves System, CNS). Therefore BBB has become a hot topic in the field of medical research. One point, especially the study of the regulation of BBB permeability, is of great significance for human understanding of the pathogenesis and treatment of brain diseases. At present, the study of pathological factors leading to changes in BBB permeability is concentrated on the injury or trauma in the brain, such as traumatic brain injury (Traumatic Brain Injury, TBI), and stroke (S Troke), ischemia (Ischemia) and so on. But the causes and mechanisms of the effects of peripheral trauma, such as surgery, and trauma, on BBB permeability are not clear. TNF-a neutralization antibody in peripheral circulation can protect the integrity of BBB. Our previous study also found that the blood brain barrier permeability increased with age (9 months and 18 months) after peripheral surgery (abdominal surgery), and the behavioral changes were changed, and the changes in the elderly group (18 months) were more obvious. These studies have shown that peripheral circulation inflammation has an impact on BBB permeability after surgical trauma. Therefore, the key to the prevention of postoperative cognitive impairment (Postoperate Cognitive Dysfunction, POCD) is the key to the prevention of postoperative cognitive impairment (Postoperate Cognitive Dysfunction, POCD). Based on this, the study is divided into the following three parts: 1. In the mouse operation model, the factors that can cause the increase of blood brain barrier permeability (anaesthesia and the inflammatory factors in the peripheral circulation) and the appropriate detection methods (the time point of BBB permeability change and the selection of tracer) are screened; 2. the peripheral blood test (enzyme linked immunosorbent assay, ELISA) is used to detect the circumference caused by the operation. The concentration of inflammatory factors (IL-6) in the circulation and the brain parenchyma (IL-6), using the molecular weight of 10KDa tracer (10 KDa Tracer) Dextran10, was used to observe the changes of BBB permeability in different age groups by fluorescence microscopy, and the degree of BBB leakage was quantified. Western Blotting method was used to detect the key protein of BBB integrity. The changes in the close connection, Tight Junction, TJ), using the Y maze (YMaze) and other behavioral tests to observe the behavioral changes in mice; 3. the use of the interleukin -6 neutralization antibody (Anti-IL-6 Neutralizing Antibody) and the use of the interleukins -6 gene knockout mice (IL-6 Gene) to verify the increase in the permeability of the key inflammatory factors The experimental results showed that 1. in the model of abdominal surgery under general anesthesia, the peripheral inflammatory factor IL-6 reached the peak at about 6 hours after the operation, and the elderly group (18 months) increased more than the normal adult group (9 months), while the serum level of IL-6 and the permeability of BBB in the anesthetic group were only given in the anesthetic group for 24 hours. There was no obvious change in sex; in the selection of tracers, only the Dextran of 10KDa, which was smaller in molecular weight, leaked out of the cerebral vessels about 6 hours after the operation, while the larger molecular weight 70KDa Dextran and Evans blue (Evans Blue, EB) did not leak out; 2. to the quantitative test of Dextran 10, the leakage of the adult operation group was about 1.3 times that of the control group. In the elderly operation group, the operation group was about 1.6 times, and the behavioral test found that there was a decrease in spatial memory in both the adult and the elderly group, and the older group was more obvious. 3. the BBB permeability of the IL-6 neutralization antibody and the IL-6 gene knockout mice before operation was not changed. The results showed that we knew the peripheral circulation inflammatory factors after the body was traumatized. It provides a theoretical basis and experimental basis for the increase of BBB permeability, and also provides a potential treatment for the prevention and treatment of complications of the central nervous system caused by peripheral trauma.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R641
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