手術(shù)創(chuàng)傷后外周循環(huán)炎癥因子對(duì)不同年齡小鼠血腦屏障通透性的影響
發(fā)布時(shí)間：2018-08-04 19:22
【摘要】：血腦屏障(Blood Brain Barrier, BBB)是存在于腦組織與血液之間復(fù)雜的細(xì)胞系統(tǒng),能阻擋病原體和有害物質(zhì)由血液進(jìn)入腦室和腦組織,是機(jī)體天然免疫的重要屏障之一。BBB主要由腦部的微血管內(nèi)皮細(xì)胞(Brain Microvascular Endothelial Cells,BMEC)、周皮細(xì)胞(Pericyte)、基膜(Basement Membrance, BM)、星形膠質(zhì)細(xì)胞(Astrocyte)和部分神經(jīng)元軸突末梢構(gòu)成。任何組成成分的破壞都將導(dǎo)致BBB通透性的變化,繼而影響中樞神經(jīng)系統(tǒng)(Central Nerves System, CNS)的健康和功能。因此BBB已成為當(dāng)今醫(yī)學(xué)研究領(lǐng)域熱點(diǎn)之一,特別是BBB通透性調(diào)控方面的研究對(duì)于人類深入認(rèn)識(shí)腦類疾病的致病機(jī)制及其治療具有重大意義。目前國(guó)內(nèi)外對(duì)病理因素導(dǎo)致BBB通透性變化的研究都集中于大腦內(nèi)部的損傷或創(chuàng)傷,如創(chuàng)傷性腦損傷(Traumatic Brain Injury, TBI),中風(fēng)(Stroke),缺血(Ischemia)等。而外周創(chuàng)傷,如手術(shù),燒創(chuàng)傷等對(duì)BBB通透性影響的原因和機(jī)制并不明確。以往研究多認(rèn)為,外周創(chuàng)傷后的應(yīng)激反應(yīng)造成中樞神經(jīng)系統(tǒng)的炎癥,進(jìn)而破壞BBB的完整性.Mervyn等2010年發(fā)表在PNAS上的研究證明術(shù)前給予小鼠外周循環(huán)注射TNF-a中和抗體可以保護(hù)BBB的完整性.我們的前期研究也發(fā)現(xiàn),不同年齡組小鼠(9個(gè)月和18個(gè)月)在經(jīng)歷外周手術(shù)(腹部手術(shù))后,血腦屏障通透性隨年齡的增長(zhǎng)有增加的趨勢(shì),且行為學(xué)發(fā)生了改變,老年組(18個(gè)月)的變化更為明顯。以上研究均表明,外周循環(huán)炎癥對(duì)手術(shù)創(chuàng)傷后BBB通透性存在著影響。因此,尋找出外周創(chuàng)傷導(dǎo)致BBB通透性增高的機(jī)制是預(yù)防臨床術(shù)后認(rèn)知障礙(Postoperate Cognitive Dysfunction, POCD)的關(guān)鍵�；诖�,本研究分為以下三部分：1.對(duì)本研究采用的小鼠手術(shù)模型中可造成血腦屏障通透性增高的因素(麻醉和外周循環(huán)中的炎癥因子)以及適當(dāng)檢測(cè)方法(BBB通透性變化的時(shí)間點(diǎn)和示蹤劑的選擇)進(jìn)行篩選；2.用酶聯(lián)免疫吸附試驗(yàn)(enzyme linked immunosorbent assay,ELISA)檢測(cè)由手術(shù)引起的外周循環(huán)中和大腦實(shí)質(zhì)組織炎癥因子(IL-6)的濃度,使用分子量為10KDa的示蹤劑(10 KDa Tracer)Dextran10,用免疫熒光的方法在熒光顯微鏡下觀察不同年齡組BBB通透性的變化,并對(duì)BBB漏出程度予以定量,使用Western Blotting方法檢測(cè)BBB完整性關(guān)鍵蛋白(緊密連接,Tight Junction,TJ)的變化,使用Y迷宮(YMaze)等行為學(xué)檢測(cè)方法觀察小鼠行為學(xué)的改變；3.使用白介素-6中和抗體(Anti-IL-6 Neutralizing Antibody)以及使用白介素-6基因敲除小鼠(IL-6 Gene Knockout mice)來(lái)驗(yàn)證關(guān)鍵炎癥因子導(dǎo)致BBB通透性增高的理論。實(shí)驗(yàn)結(jié)果顯示：1.本研究小鼠在全麻狀態(tài)下行腹部手術(shù)的模型中,外周炎癥因子IL-6在手術(shù)后6小時(shí)左右達(dá)到高峰,且老年組(18個(gè)月)比普通成年組(9個(gè)月)上升幅度更大,而僅給予麻醉組小鼠在24小時(shí)內(nèi)血清IL-6水平以及BBB的通透性沒(méi)有明顯變化；在示蹤劑的選擇上,僅觀察到分子量較小的10KDa的Dextran于在術(shù)后6小時(shí)左右由腦血管漏出,而分子量較大的70KDa Dextran和伊文氏藍(lán)(Evans Blue,EB)沒(méi)有漏出；2.對(duì)Dextran 10的定量實(shí)驗(yàn)中,成年手術(shù)組漏出量約為對(duì)照組的1.3倍,而老年手術(shù)組為1.6倍左右;行為學(xué)檢測(cè)發(fā)現(xiàn)術(shù)后成年組和老年組小鼠均出現(xiàn)空間記憶力的下降,老年組更為明顯；3.術(shù)前使用IL-6中和抗體以及IL-6基因敲除鼠的BBB通透性沒(méi)有變化。以上結(jié)果為我們認(rèn)識(shí)機(jī)體創(chuàng)傷后,外周循環(huán)炎癥因子導(dǎo)致BBB通透性增加提供了理論依據(jù)和實(shí)驗(yàn)基礎(chǔ),也為臨床上防治由外周創(chuàng)傷引起的中樞神經(jīng)系統(tǒng)并發(fā)癥提供了潛在的治療方法。
[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.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：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.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R641
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