RNA干擾水通道蛋白4對(duì)創(chuàng)傷性腦水腫血腦屏障保護(hù)作用的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-10-22 16:59
【摘要】：創(chuàng)傷后腦水腫是創(chuàng)傷性腦損傷（traumatic brain injury，TBI）后最重要的繼發(fā)性病理生理反應(yīng)，其病理反應(yīng)為過(guò)多的水分積聚在細(xì)胞內(nèi)外間隙，導(dǎo)致腦體積增大，是影響傷情及預(yù)后的重要因素之一。臨床上創(chuàng)傷性腦水腫的主要危害是升高顱內(nèi)壓、壓迫腦組織甚至形成腦疝，為導(dǎo)致死亡和病殘的主要原因之一。目前創(chuàng)傷性腦水腫的發(fā)生機(jī)理仍不十分明確，綜合各種觀點(diǎn)有如下學(xué)說(shuō)：⑴血腦屏障破（blood-brain barrier，BBB）壞學(xué)說(shuō)；⑵細(xì)胞內(nèi)鈣超載學(xué)說(shuō)；⑶自由基損傷學(xué)說(shuō)；⑷腦血液流變學(xué)變化和腦微循環(huán)障礙學(xué)說(shuō)；⑸細(xì)胞能量代謝紊亂學(xué)說(shuō);⑹創(chuàng)傷性炎癥反應(yīng)學(xué)說(shuō)。BBB損害為血管源性腦水腫的病理基礎(chǔ)，缺血缺氧引起的BBB結(jié)構(gòu)和功能改變可能是導(dǎo)致TBI后腦水腫的最早和最重要的原因。然而目前為止，腦創(chuàng)傷早期BBB改變的分子生物學(xué)機(jī)制尚不清楚，并且，在臨床上仍然缺乏阻止創(chuàng)傷性腦水腫發(fā)生和發(fā)展的有效藥物。目前腦水腫治療上主要局限于滲透性利尿、高張鹽水及外科減壓等。針對(duì)抑制腦水腫形成及發(fā)展方面的藥物尚未見報(bào)道。水通道蛋白(aquaporins，，AQPs)的發(fā)現(xiàn)為研究一種副作用小且適用于各種類型腦水腫的治療藥物提供了可能。 AQPs是一個(gè)高選擇性、低活化能轉(zhuǎn)運(yùn)水分子的跨膜通道蛋白家族。AQP4是中樞神經(jīng)系統(tǒng)分部最廣，也是最重要的AQPs。AQP4主要分布于軟腦膜，導(dǎo)管系統(tǒng)的室管膜、腦室、脈絡(luò)叢等與腦脊液直接相接觸的組織和腦實(shí)質(zhì)的血管周圍，尤其在與軟腦膜和毛細(xì)血管接觸面直接相連的星形膠質(zhì)細(xì)胞足突上表達(dá)特別豐富。AQP4有細(xì)胞外壓力感受器和水平衡調(diào)節(jié)功能，是介導(dǎo)腦組織水與電解質(zhì)運(yùn)輸和平衡的重要因素。國(guó)外有人運(yùn)用siRNA干擾方法,轉(zhuǎn)染體外培養(yǎng)的星形膠質(zhì)細(xì)胞，使AQP4和mRNA的表達(dá)減少，國(guó)內(nèi)也有人應(yīng)用質(zhì)粒做過(guò)相似的實(shí)驗(yàn)，同樣取得良好的效果。AQP4基因敲除小鼠創(chuàng)傷性和水中毒性腦水腫的程度明顯減輕。文獻(xiàn)檢索證實(shí)我省還沒有對(duì)該領(lǐng)域進(jìn)行研究。我們擬對(duì)AQP4進(jìn)行RNA干擾（RNAinterference，RNAi），判斷其對(duì)創(chuàng)傷后腦水腫的抑制效果及對(duì)BBB的保護(hù)作用。 目的 探討RNA干擾AQP4對(duì)創(chuàng)傷性腦水腫BBB的保護(hù)作用。 方法 健康成年雄性wistar大鼠264只,體質(zhì)量控制在250-300g之間，參照Marmarou法制作TBI模型，TBI后在腦立體定向儀輔助下向腦室內(nèi)注入AQP4RNAi質(zhì)粒、空白對(duì)照質(zhì)�；蛸|(zhì)粒溶劑，以判斷RNAi對(duì)腦水腫的治療作用及對(duì)BBB的保護(hù)作用。 主要采用以下實(shí)驗(yàn)方法進(jìn)行相關(guān)研究：一、干濕重法測(cè)腦組織含水量 腦組織稱取濕重后放入電熱烘箱內(nèi)100℃烘烤24h，再稱干重，按如下公式計(jì)算腦組織含水量。腦組織含水量=（濕重-干重）/濕重×100%。二、EB測(cè)定BBB通透性改變 利用尾靜脈注射EB溶液，取損傷區(qū)腦組織檢測(cè)EB含量，作為評(píng)價(jià)BBB通透性的指標(biāo)，分析判定BBB通透性的變化趨勢(shì)。三、AQP4mRNA原位雜交觀察 利用原位雜交法鑒定AQP4mRNA在各實(shí)驗(yàn)處理組情況下?lián)p傷中心腦組織表達(dá)情況。并通過(guò)Image-proplus6.0圖像分析軟件計(jì)算全腦單位面積AQP4mRNA平均陽(yáng)性強(qiáng)度。四、AQP4、ZO-1免疫熒光組織化學(xué)觀察 利用免疫熒光組織化學(xué)檢測(cè)在各實(shí)驗(yàn)處理組情況下AQP4的表達(dá)情況，分析判斷TBI后AQP4的表達(dá)變化情況和腦水腫發(fā)生發(fā)展的相關(guān)性及鑒定干擾效果；檢測(cè)ZO-1表達(dá)變化情況，分析和印證各實(shí)驗(yàn)組BBB通透性變化。五、光電鏡觀察細(xì)胞形態(tài)學(xué)變化及超微結(jié)構(gòu)改變 各實(shí)驗(yàn)組大鼠分時(shí)間點(diǎn)，制作石蠟切片及超薄切片，分別用普通光學(xué)顯微鏡及透射電子顯微鏡進(jìn)行觀察。觀測(cè)各實(shí)驗(yàn)處理后大鼠腦組織細(xì)胞形態(tài)學(xué)改變和BBB結(jié)構(gòu)變化及超微結(jié)構(gòu)改變。六、統(tǒng)計(jì)學(xué)分析 SPSS17.0版統(tǒng)計(jì)分析軟件用于分析數(shù)據(jù)。根據(jù)數(shù)據(jù)的性質(zhì)選取單因素方差分析或Pearson相關(guān)系數(shù)等分析方法。P值小于0.05視為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果 RNAi質(zhì)�？捎行p少AQP4在受損腦組織的表達(dá)；RNAi組在各檢測(cè)時(shí)間點(diǎn)腦組織含水量和BBB通透性均小于TBI組和對(duì)照質(zhì)粒組(P0.05)；TBI后RNAi組ZO-1表達(dá)水平各時(shí)間點(diǎn)明顯高于單純創(chuàng)傷組和對(duì)照粒組（P＜0.05）；光鏡下可見：RNAi組大鼠星形膠質(zhì)細(xì)胞和神經(jīng)元水腫的范圍和程度均低于單純創(chuàng)傷組和對(duì)照質(zhì)粒組；假手術(shù)組膠質(zhì)細(xì)胞和神經(jīng)元未見明顯的水腫。透射電鏡下可見：RNAi組大鼠超微結(jié)構(gòu)改變、緊密連接開放程度均低于單純創(chuàng)傷組和對(duì)照質(zhì)粒組；假手術(shù)組超微結(jié)構(gòu)改變和緊密連接（tight junction，TJ）未見明顯的改變。 結(jié)論 顱腦創(chuàng)傷后AQP4蛋白的表達(dá)變化趨勢(shì)和腦水腫的發(fā)展趨勢(shì)相一致，二者緊密相關(guān)；應(yīng)用RNA干擾方法能有效減少AQP4的表達(dá)，可有效減少TBI后腦水腫BBB結(jié)構(gòu)的破壞，對(duì)BBB起保護(hù)作用。
[Abstract]:Post-traumatic brain edema is the most important secondary pathological change after traumatic brain injury (TBI). The pathological response of traumatic brain edema is one of the important factors influencing the injury and prognosis. The main harm of clinical traumatic brain edema is to raise intracranial pressure, compress brain tissue and even form cerebral hernia, which is one of the main causes of death and disability. At present, the mechanism of traumatic brain edema is still not very clear, and the comprehensive opinions are as follows: blood-brain barrier (BBB) bad theory; the theory of calcium overload in human brain cells; the theory of free radical damage; the changes of hemorheology and the theory of cerebral microcirculation disturbance; The theory of energy metabolism disorder in nuclear fission cells; the theory of traumatic inflammation in rats. The change of vascular structure and function caused by ischemia and hypoxia may be the earliest and most important cause of brain edema after TBI. Up to now, the mechanism of molecular biology that has changed early in brain trauma is unknown, and there is still a lack of effective drugs to prevent the occurrence and development of traumatic brain edema. At present, the treatment of cerebral edema is mainly limited to osmotic diuresis, hypertonic saline and surgical decompression. Drugs that inhibit the formation and development of brain edema have not yet been reported. The discovery of aquaporin, AQPs provides the possibility to study a therapeutic drug that is small in side effect and suitable for various types of brain edema. AQPs is a transmembrane channel protein with high selectivity and low activation energy transfer water molecules The AQP4 is the most extensive and most important AQPs in the central nervous system. AQP4 is mainly distributed in the soft meninges, the inner wall of the catheter system, the ventricle, the nerve plexus and the like, and the parenchyma and the parenchyma of the brain which are directly in contact with the cerebrospinal fluid. Peripheral, especially in astrocytes that are directly connected to the contact surfaces of the meninges and capillary vessels. Rich. AQP4 has extracellular pressure receptor and water balance regulating function, which is an important role in mediating the transportation and balance of water and electrolyte in brain tissue. The results showed that the expression of AQP4 and mRNA was reduced by using siRNA interference method, and the expression of AQP4 and mRNA was decreased. The effects of AQP4 gene knockout mice in traumatic and water toxic brain edema were obvious. Mitigation. The literature search confirms that our province has not yet done so To study the effect of RNA interference (RNAi) on AQP4 and its protective effect on post-traumatic brain edema. function Objective To investigate the effect of RNA interference AQP4 on traumatic brain edema BB B. Methods Healthy adult male Wistar rats were divided into 264 rats with mass control of 250-300g. The TBI model was made by Marmaru method. After TBI, AQP4RNAi was injected into the brain under the aid of stereotactic instrument. plasmid, blank control plasmid or plasmid solvent to judge the effect of RNAi on cerebral edema The treatment effect and the protective effect on the liver cancer are mainly carried out by the following experimental methods: Related research: First, wet and dry weight method to test the brain tissue water content of brain tissue, weigh the wet weight, put into the electric heating oven to bake at 100 鈩
本文編號(hào)：2287761
[Abstract]:Post-traumatic brain edema is the most important secondary pathological change after traumatic brain injury (TBI). The pathological response of traumatic brain edema is one of the important factors influencing the injury and prognosis. The main harm of clinical traumatic brain edema is to raise intracranial pressure, compress brain tissue and even form cerebral hernia, which is one of the main causes of death and disability. At present, the mechanism of traumatic brain edema is still not very clear, and the comprehensive opinions are as follows: blood-brain barrier (BBB) bad theory; the theory of calcium overload in human brain cells; the theory of free radical damage; the changes of hemorheology and the theory of cerebral microcirculation disturbance; The theory of energy metabolism disorder in nuclear fission cells; the theory of traumatic inflammation in rats. The change of vascular structure and function caused by ischemia and hypoxia may be the earliest and most important cause of brain edema after TBI. Up to now, the mechanism of molecular biology that has changed early in brain trauma is unknown, and there is still a lack of effective drugs to prevent the occurrence and development of traumatic brain edema. At present, the treatment of cerebral edema is mainly limited to osmotic diuresis, hypertonic saline and surgical decompression. Drugs that inhibit the formation and development of brain edema have not yet been reported. The discovery of aquaporin, AQPs provides the possibility to study a therapeutic drug that is small in side effect and suitable for various types of brain edema. AQPs is a transmembrane channel protein with high selectivity and low activation energy transfer water molecules The AQP4 is the most extensive and most important AQPs in the central nervous system. AQP4 is mainly distributed in the soft meninges, the inner wall of the catheter system, the ventricle, the nerve plexus and the like, and the parenchyma and the parenchyma of the brain which are directly in contact with the cerebrospinal fluid. Peripheral, especially in astrocytes that are directly connected to the contact surfaces of the meninges and capillary vessels. Rich. AQP4 has extracellular pressure receptor and water balance regulating function, which is an important role in mediating the transportation and balance of water and electrolyte in brain tissue. The results showed that the expression of AQP4 and mRNA was reduced by using siRNA interference method, and the expression of AQP4 and mRNA was decreased. The effects of AQP4 gene knockout mice in traumatic and water toxic brain edema were obvious. Mitigation. The literature search confirms that our province has not yet done so To study the effect of RNA interference (RNAi) on AQP4 and its protective effect on post-traumatic brain edema. function Objective To investigate the effect of RNA interference AQP4 on traumatic brain edema BB B. Methods Healthy adult male Wistar rats were divided into 264 rats with mass control of 250-300g. The TBI model was made by Marmaru method. After TBI, AQP4RNAi was injected into the brain under the aid of stereotactic instrument. plasmid, blank control plasmid or plasmid solvent to judge the effect of RNAi on cerebral edema The treatment effect and the protective effect on the liver cancer are mainly carried out by the following experimental methods: Related research: First, wet and dry weight method to test the brain tissue water content of brain tissue, weigh the wet weight, put into the electric heating oven to bake at 100 鈩
本文編號(hào)：2287761
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