【摘要】： 目前認(rèn)為，各種腦損傷(腦缺血、腦外傷)的繼發(fā)性損傷，或稱二次腦損傷因素，是造成腦損害發(fā)生、發(fā)展的重要原因。這些因素涉及：腦缺血、能量代謝障礙、鈣離子超載、氧自由基堆積、興奮性氨基酸的神經(jīng)毒作用、炎性因子刺激等。其中，炎癥是腦損傷病理生理過程中的一個重要組成環(huán)節(jié)，半胱氨酰白三烯(cysteinyl leukotrienes, CysLTs，包括LTC_4、LTD_4和LTE_4)是一類重要的炎癥介質(zhì)，是花生四烯酸5-脂氧合酶(5-lipoxygenase, 5-LOX)的代謝產(chǎn)物。CysLTs通過激活半胱氨酰白三烯受體(包括CysLT_1和CysLT_2受體)，參與多種炎癥病理過程，介導(dǎo)平滑肌痙攣、微血管滲漏等反應(yīng)。近年證明，除了在哮喘、過敏性鼻炎等炎癥疾病發(fā)病過程中起重要作用外，CysLTs也參與腦卒中、腦外傷、腦腫瘤、癲癇等中樞神經(jīng)系統(tǒng)疾病的病理生理過程。腦缺血后CysLTs釋放增加，5-LOX抑制劑能減少CysLTs產(chǎn)生，保護(hù)腦缺血性損傷，本實驗室也發(fā)現(xiàn)，CysLT_1受體拮抗劑普魯司特和孟魯司特對實驗性腦缺血損傷有保護(hù)作用，普魯司特對NMDA引起的皮層損傷及小鼠腦冷凍傷也有保護(hù)作用。這些發(fā)現(xiàn)均提示CysLTs參與腦損傷病理過程，然而，CysLTs對神經(jīng)元是否有直接致?lián)p傷作用?這種作用通過哪種受體亞型介導(dǎo)?這些問題仍有待闡明。 腦損傷是高度復(fù)雜的病理過程，而腦水腫是各種腦損傷的共同病理環(huán)節(jié)。腦水腫一般包括血管性腦水腫和細(xì)胞性腦水腫。血管性腦水腫(vasogenic brainedema)是由于血腦屏障通透性增高，，導(dǎo)致血漿蛋白滲漏到細(xì)胞間隙，造成細(xì)胞間隙膠體滲透壓增高而導(dǎo)致腦組織腫脹；細(xì)胞性腦水腫(cytotoxic brain edema)是由于細(xì)胞能量代謝障礙，導(dǎo)致離子泵(如Na~+-K~+-ATP酶等)轉(zhuǎn)運功能失調(diào)，離子蓄積在細(xì)胞內(nèi)而導(dǎo)致細(xì)胞腫脹。腦水腫形成的根本原因，在于水分子跨細(xì)胞膜轉(zhuǎn)運功能障礙，這種障礙涉及水通道蛋白(aquaporins,AQPs)。 AQPs是一組介導(dǎo)水分子跨膜轉(zhuǎn)運的細(xì)胞膜蛋白，AQP4是腦內(nèi)最主要的AQPs亞型。已經(jīng)發(fā)現(xiàn)，包括腦外傷、腦缺血、腦腫瘤等多種腦損傷病理中，伴隨腦內(nèi)AQP4表達(dá)上調(diào)，而且與腦水腫發(fā)生密切相關(guān)，對AQP4基因敲除動物的研究結(jié)果提示，AQP4對血管性腦水腫和細(xì)胞性腦水腫的形成均有影響。在我們的前期工作中，以藥理學(xué)方法證實CysLTs／CysLT受體參與缺血性腦損傷，其機制與血腦屏障(BBB)破壞、腦水腫發(fā)生相關(guān)，但是，在腦水腫形成過程中CysLTs與腦內(nèi)AQP4間是否存在聯(lián)系，以及與AQP4表達(dá)調(diào)節(jié)有關(guān)的CysLT受體亞型等問題，有待于進(jìn)一步闡明。 為深入探討CysLTs對腦的直接致?lián)p傷作用，本研究建立CysLTs小鼠皮層注射模型，考察CysLTs對血腦屏障功能、腦水腫的影響，以及CysLTs引起的神經(jīng)元變性、死亡和腦梗死等作用；同時，觀察CysLT_1受體拮抗劑普魯司特對CysLTs致腦損傷作用的影響。我們還建立了大鼠原代星形膠質(zhì)細(xì)胞LTD_4損傷模型，考察LTD_4對AQP4表達(dá)的調(diào)節(jié)作用；同時，以普魯司特和CysLT_1／CysLT_2受體非選擇性拮抗劑BAYu9773干預(yù)，以了解與該作用有關(guān)的CysLT受體亞型。 第—部分 外源性半胱氨酰白三烯對小鼠腦皮層的致?lián)p傷作用 腦損傷引起腦內(nèi)CysLTs釋放增加，CysLT受體誘導(dǎo)表達(dá)；5-LOX抑制劑能減少CysLTs產(chǎn)生，減輕腦缺血性損傷；CysLT_1受體拮抗劑普魯司特和孟魯司特，也可減輕實驗性腦缺血損傷。這些結(jié)果均表明，CysLTs參與腦損傷過程。為進(jìn)一步探討CysLTs對腦的直接致?lián)p傷作用及其特征，了解與CysLTs致腦損傷作用有關(guān)的受體亞型，我們研究了CysLTs(LTC_4、LTD_4)小鼠皮層注射致腦損傷的量效關(guān)系和時效關(guān)系，同時觀察了CysLTs引起的BBB功能損害、腦水腫形成、神經(jīng)元變性、死亡，以及對皮層AQP4表達(dá)的影響。結(jié)果顯示，小鼠皮層注射LTC_4、LTD_4(＞1ng/0.5μl PBS)均可造成皮層損傷；LTD_4(1ng/0.5μl PBS)造成的皮層損傷，發(fā)生于注射后6小時，伴隨皮層內(nèi)源性IgG滲出增加，腦水腫形成，同時出現(xiàn)Fluoro-Jade B染色陽性細(xì)胞(變性神經(jīng)元)數(shù)量增加，存活神經(jīng)元計數(shù)減少，以及皮層AQP4表達(dá)上調(diào)。CysLT_1受體拮抗劑普魯司特，能減小LTD_4引起的皮層梗死面積，抑制皮層內(nèi)源性IgG滲出及神經(jīng)元變性、死亡；但是，不影響LTD_4引起的腦水腫和皮層AQP4表達(dá)上調(diào)。結(jié)果表明，CysLTs對小鼠皮層具有致?lián)p傷作用，其機制與BBB破壞、腦水腫發(fā)生相關(guān)；CysLTs通過CysLT_1受體介導(dǎo)BBB開放，引起血管性腦水腫，導(dǎo)致腦損傷；CysLTs還可能通過上調(diào)AQP4表達(dá)引起細(xì)胞性腦水腫，而該作用并非由CysLT_1受體介導(dǎo)。 第二部分 白三烯D_4對大鼠原代星形膠質(zhì)細(xì)胞水通道蛋白4的調(diào)節(jié)作用 腦內(nèi)水平衡與水通道蛋白(aquaporins，AQPs)有關(guān)。其中，AQP4是腦內(nèi)最重要的AQP亞型，AQP4主要分布在腦內(nèi)星形膠質(zhì)細(xì)胞及其突起，也表達(dá)在室管膜細(xì)胞以及腦血管內(nèi)皮細(xì)胞，是調(diào)節(jié)腦內(nèi)水平衡的一種主要機制。腦損傷(腦缺血、腦外傷、腦腫瘤和腦水中毒)可以引起AQP4表達(dá)改變，與腦水腫形成密切相關(guān)。AQP4基因缺失可以減輕局灶性腦缺血、腦水中毒及細(xì)菌性腦膜炎引起的細(xì)胞性水腫，但可促進(jìn)腦腫瘤、腦膿腫，皮層冷凍傷和腦積水引起的血管性水腫形成；提示AQP4對腦血管性水腫具有抑制作用，但促進(jìn)細(xì)胞性水腫形成。在第一部分研究中，我們發(fā)現(xiàn)LTD_4引起的腦水腫伴隨皮層AQP4表達(dá)增加，CysLT_1受體拮抗劑普魯司特不影響該作用。為了驗證CysLTs對AQP4表達(dá)的調(diào)節(jié)作用，闡明與該作用有關(guān)的CysLT受體亞型，我們建立了大鼠原代星形膠質(zhì)細(xì)胞LTD_4損傷模型。結(jié)果發(fā)現(xiàn)，LTD_4(10~(-9)-10~(-7)mol／L)使體外培養(yǎng)的大鼠原代星形膠質(zhì)細(xì)胞AQP4表達(dá)上調(diào)，同時伴隨細(xì)胞形態(tài)改變，作用具有濃度依賴性。研究還發(fā)現(xiàn)，大鼠原代星形膠質(zhì)細(xì)胞僅有微量CysLT_2受體mRNA表達(dá)，而CysLT_1受體mRNA表達(dá)相對較高，LTD_4(10~(-9)-10~(-7)mol／L)對星形膠質(zhì)細(xì)胞CysLT_1受體mRNA表達(dá)無顯著影響，但可濃度依賴性上調(diào)CysLT_2受體mRNA表達(dá)。選擇性CysLT_1受體拮抗劑普魯司特、孟魯司特，對LTD_4(10~(-9)-10~(-7)mol／L)引起的AQP4表達(dá)上調(diào)無顯著影響；CysLT_1／CysLT_2受體非選擇性拮抗劑BAYu9773，顯著抑制LTD_4(10~(-9)-10~(-7)mol／L)引起的AQP4表達(dá)上調(diào)。這些結(jié)果提示，LTD_4可上調(diào)AQP4表達(dá)，并誘導(dǎo)表達(dá)星形膠質(zhì)細(xì)胞CysLT_2受體；提示炎癥過程中釋放的CysLTs可通過CysLT_2受體促進(jìn)細(xì)胞性腦水腫形成，這可能是CysLTs致腦損傷作用的另一機制。 總結(jié) 1、外源性CysLTs(LTC_4、LTD_4)對小鼠腦皮層具有時間和劑量依賴的致?lián)p傷作用，其機制與血腦屏障破壞相關(guān)。CysLT_1受體拮抗劑普魯司特對CysLTs引起的血腦屏障破壞和皮層損傷具有保護(hù)作用，表明CysLTs通過CysLT_1受體誘導(dǎo)血管性腦水腫，導(dǎo)致腦損傷。 2、外源性LTD_4引起小鼠腦皮層AQP4表達(dá)上調(diào)和腦水腫；普魯司特對LTD_4引起的AQP4表達(dá)上調(diào)和腦水腫無抑制作用。 3、外源性LTD_4濃度依賴性上調(diào)大鼠原代星形膠質(zhì)細(xì)胞AQP4表達(dá)；CysLT_1受體拮抗劑普魯司特、孟魯司特對LTD_4引起的AQP4表達(dá)上調(diào)無明顯作用，但CysLT受體非選擇性拮抗劑BAYu9773可阻斷這種上調(diào)；LTD_4還可濃度依賴性上調(diào)CysLT_2受體mRNA表達(dá)。表明LTD_4可通過激動CysLT_2受體調(diào)節(jié)AQP4表達(dá)，引起細(xì)胞性腦水腫。
[Abstract]:It is believed that the secondary injury of brain injury (cerebral ischemia, brain injury), or two times of brain injury, is an important cause of the development of brain damage. These factors include cerebral ischemia, energy metabolism disorder, calcium overload, oxygen free radical accumulation, stimulating amino acid neurotoxicity, inflammatory factor stimulation and so on. Disease is an important component of the pathophysiological process of brain injury. Cysteinyl leukotrienes (CysLTs, including LTC_4, LTD_4 and LTE_4) is an important class of inflammatory mediators, and is a metabolite of the 5- lipoxygenase (5-lipoxygenase, 5-LOX) of peanut four, which activates the cysteinyl leukotriene receptor (including Cy) by activating the receptor of the cysteinyl leukotrienes (5-lipoxygenase, 5-LOX). SLT_1 and CysLT_2 receptor), involved in a variety of inflammatory pathological processes, mediating smooth muscle spasm, microvascular leakage and other reactions. In recent years, in addition to the important role of asthma, allergic rhinitis and other inflammatory diseases, CysLTs also participates in the pathophysiological process of cerebral apoplexy, brain trauma, brain swelling, epilepsy and other central nervous system diseases. CysLTs release increases after ischemia, and 5-LOX inhibitors can reduce CysLTs production and protect ischemic brain damage. This laboratory also found that the CysLT_1 receptor antagonist pruscit and montelukast have protective effects on experimental cerebral ischemia injury. Prusxate also protects the cortex injury caused by NMDA and the frozen brain injury in mice. It is suggested that CysLTs is involved in the pathological process of brain injury. However, does CysLTs have direct damage to neurons? Which receptor subtypes are mediated by this effect? These problems remain to be elucidated.
Brain edema is a highly complex pathological process, and brain edema is a common pathological link of various brain injuries. Cerebral edema usually includes vascular cerebral edema and cellular brain edema. Vascular cerebral edema (vasogenic brainedema) is due to the increase of blood brain barrier permeability, causing plasma egg white leakage to intercellular space and intercellular colloid infiltration. The increase of pressure leads to the swelling of the brain tissue; cellular brain edema (cytotoxic brain edema) is due to the disorder of cell energy metabolism, which leads to the imbalance of the transport function of the ion pump (such as Na~+-K~+-ATP enzyme). The accumulation of ions in the cell causes the swelling of the cells. The root cause of the formation of brain edema lies in the transcellular membrane transport dysfunction of water molecules. The barrier involves aquaporins (AQPs).
AQPs is a group of cell membrane proteins that mediate transmembrane transport of water molecules. AQP4 is the most important AQPs subtype in the brain. It has been found that the expression of AQP4 in brain injury, cerebral ischemia and brain tumor is associated with the up regulation of the expression of AQP4 in the brain, and it is closely related to brain edema. The results of the study of AQP4 gene knockout animals suggest that AQP4 is on the blood vessels. In our previous work, the mechanism of CysLTs / CysLT receptor involved in ischemic brain injury was related to the destruction of the blood brain barrier (BBB) and the occurrence of brain edema in our previous work. However, there was a relationship between CysLTs and AQP4 in the formation of brain edema, and with AQP4. The expression of CysLT receptor subtypes needs further clarification.
In order to investigate the direct damage of CysLTs to the brain, this study established an injection model of CysLTs mice to investigate the effect of CysLTs on the function of blood brain barrier and brain edema, as well as the effects of CysLTs induced degeneration, death and cerebral infarction. At the same time, the effect of CysLT_1 receptor antagonist prusst on CysLTs induced brain damage was observed. We also established the LTD_4 damage model of rat primary astrocytes to investigate the role of LTD_4 in the regulation of AQP4 expression; at the same time, the CysLT receptor subtype related to this effect was detected by the intervention of Prussian and CysLT_1 / CysLT_2 receptor non selective antagonist BAYu9773.
Part - Part
Effects of exogenous cysteinyl leukotriene on cerebral cortex in mice
Brain injury causes increased CysLTs release in the brain, induced expression of CysLT receptor, 5-LOX inhibitor can reduce CysLTs production and reduce cerebral ischemic damage; CysLT_1 receptor antagonist pruscit and montelukast can also reduce experimental cerebral ischemia injury. These results suggest that CysLTs and brain damage process. To further explore CysLTs to the brain Direct injury and its characteristics, and understanding of the receptor subtypes associated with brain damage induced by CysLTs, we studied the dose effect relationship and aging relationship of cerebral injury induced by cortical injection of CysLTs (LTC_4, LTD_4) mice, and observed the BBB function damage caused by CysLTs, the formation of brain edema, neuron degeneration, death, and the expression of AQP4 in the cortex of the cortex. The results showed that the cortical injection of LTC_4, LTD_4 (> 1ng/0.5 Mu L PBS) in mice could cause cortical damage, and the cortical injury caused by LTD_4 (1ng/0.5 Mu L PBS) occurred at 6 hours after the injection, accompanied by the increase of endogenous IgG exudation in the cortex, the formation of brain edema, and the increase in the number of Fluoro-Jade B staining positive cells (denatured neurons). The decrease of the cell count and the up-regulation of.CysLT_1 receptor antagonist prushia, the expression of AQP4, can reduce the area of cortical infarct caused by LTD_4, inhibit the endogenous IgG exudation and degeneration of the cortex, and die. However, it does not affect the brain edema caused by LTD_4 and the expression of AQP4 in the cortex. The results show that CysLTs has a damage to the cortex of the mouse. The mechanism is related to BBB destruction and brain edema; CysLTs is open to BBB through CysLT_1 receptor, causing vascular cerebral edema and causing brain damage; CysLTs may also induce cellular brain edema by up regulation of AQP4 expression, and this effect is not mediated by CysLT_1 receptor.
The second part
Regulatory effect of leukotriene D_4 on aquaporin 4 in rat primary astrocytes
Brain water balance is related to aquaporins (AQPs). Among them, AQP4 is the most important AQP subtype in the brain. AQP4 is mainly distributed in astrocytes and their protrusions in the brain. It is also expressed in ependymal cells and cerebral vascular endothelial cells. It is a major mechanism for regulating the balance of water in the brain. Brain injury (cerebral ischemia, brain injury, brain tumor and brain tumor) Cerebral water intoxication can cause changes in AQP4 expression..AQP4 gene deletion closely related to brain edema can reduce focal cerebral ischemia, cerebral water intoxication and bacterial meningitis caused by cellular edema, but can promote cerebral tumor, brain abscess, cerebral cortex freeze injury and hydrocephalus formation caused by cerebral hydrocephalus, prompting AQP4 to cerebral vascular water Swelling has a inhibitory effect, but promotes the formation of cellular edema. In the first part of the study, we found that LTD_4 induced brain edema was associated with an increase in the expression of AQP4 in the cortex, and the CysLT_1 receptor antagonist, prusthe did not affect this effect. In order to verify the regulatory role of CysLTs on AQP4 expression, we set up the CysLT receptor subtype related to this role. The results showed that LTD_4 (10~ (-9) -10~ (-7) mol / L) increased the AQP4 expression of the primary astrocytes in vitro, and accompanied the cell morphologic changes in the rat. It was found that the primary astrocytes of the rats had a concentration dependent effect. It was also found that the primary astrocytes of the rat were only the mRNA expression of the CysLT_2 receptor in the primary astrocytes. The expression of CysLT_1 receptor mRNA is relatively high, and LTD_4 (10~ (-9) -10~ (-7) mol / L) has no significant effect on mRNA expression of CysLT_1 receptor in astrocytes, but the concentration dependent up regulation of CysLT_2 receptor expression. The CysLT_1 / CysLT_2 receptor non selective antagonist, BAYu9773, significantly inhibits the up regulation of LTD_4 (10~ (-9) -10~ (-7) mol / L). These results suggest that LTD_4 can up-regulate the expression and induce the expression of astrocyte receptor. The formation of brain edema may be another mechanism of CysLTs induced brain injury.
summary
1, exogenous CysLTs (LTC_4, LTD_4) has a time and dose dependent damage to the cerebral cortex of mice. The mechanism is protected by the.CysLT_1 receptor antagonist prusk, a.CysLT_1 receptor antagonist, on the damage of blood brain barrier and cortical damage caused by CysLTs, which indicates that CysLTs can induce vascular cerebral edema through the CysLT_1 receptor. Brain damage.
2. Exogenous LTD_4 induced up-regulation of AQP4 expression in the cerebral cortex and cerebral edema in mice; Propristine did not inhibit up-regulation of AQP4 expression and brain edema induced by LTD_4.
3, exogenous LTD_4 concentration dependent up up the expression of AQP4 in rat primary astrocytes; CysLT_1 receptor antagonist pruscid and montelukast did not increase the up regulation of AQP4 expression induced by LTD_4, but CysLT receptor non selective antagonist BAYu9773 could block the up regulation, and LTD_4 also can increase the expression of CysLT_2 receptor mRNA in a concentration dependent manner. It is suggested that LTD_4 can regulate the expression of AQP4 by stimulating CysLT_2 receptor and induce cellular brain edema.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：R363

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1 王夢令;半胱氨酰白三烯對腦的致?lián)p傷作用及其機制研究[D];浙江大學(xué);2006年

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