RNA干扰水通道蛋白4对创伤性脑水肿血脑屏障保护作用的实验研究
发布时间：2018-10-22 16:59
【摘要】：创伤后脑水肿是创伤性脑损伤（traumatic brain injury，TBI）后最重要的继发性病理生理反应，其病理反应为过多的水分积聚在细胞内外间隙，导致脑体积增大，是影响伤情及预后的重要因素之一。临床上创伤性脑水肿的主要危害是升高颅内压、压迫脑组织甚至形成脑疝，为导致死亡和病残的主要原因之一。目前创伤性脑水肿的发生机理仍不十分明确，综合各种观点有如下学说：⑴血脑屏障破（blood-brain barrier，BBB）坏学说；⑵细胞内钙超载学说；⑶自由基损伤学说；⑷脑血液流变学变化和脑微循环障碍学说；⑸细胞能量代谢紊乱学说;⑹创伤性炎症反应学说。BBB损害为血管源性脑水肿的病理基础，缺血缺氧引起的BBB结构和功能改变可能是导致TBI后脑水肿的最早和最重要的原因。然而目前为止，脑创伤早期BBB改变的分子生物学机制尚不清楚，并且，在临床上仍然缺乏阻止创伤性脑水肿发生和发展的有效药物。目前脑水肿治疗上主要局限于渗透性利尿、高张盐水及外科减压等。针对抑制脑水肿形成及发展方面的药物尚未见报道。水通道蛋白(aquaporins，，AQPs)的发现为研究一种副作用小且适用于各种类型脑水肿的治疗药物提供了可能。 AQPs是一个高选择性、低活化能转运水分子的跨膜通道蛋白家族。AQP4是中枢神经系统分部最广，也是最重要的AQPs。AQP4主要分布于软脑膜，导管系统的室管膜、脑室、脉络丛等与脑脊液直接相接触的组织和脑实质的血管周围，尤其在与软脑膜和毛细血管接触面直接相连的星形胶质细胞足突上表达特别丰富。AQP4有细胞外压力感受器和水平衡调节功能，是介导脑组织水与电解质运输和平衡的重要因素。国外有人运用siRNA干扰方法,转染体外培养的星形胶质细胞，使AQP4和mRNA的表达减少，国内也有人应用质粒做过相似的实验，同样取得良好的效果。AQP4基因敲除小鼠创伤性和水中毒性脑水肿的程度明显减轻。文献检索证实我省还没有对该领域进行研究。我们拟对AQP4进行RNA干扰（RNAinterference，RNAi），判断其对创伤后脑水肿的抑制效果及对BBB的保护作用。 目的 探讨RNA干扰AQP4对创伤性脑水肿BBB的保护作用。 方法 健康成年雄性wistar大鼠264只,体质量控制在250-300g之间，参照Marmarou法制作TBI模型，TBI后在脑立体定向仪辅助下向脑室内注入AQP4RNAi质粒、空白对照质粒或质粒溶剂，以判断RNAi对脑水肿的治疗作用及对BBB的保护作用。 主要采用以下实验方法进行相关研究：一、干湿重法测脑组织含水量 脑组织称取湿重后放入电热烘箱内100℃烘烤24h，再称干重，按如下公式计算脑组织含水量。脑组织含水量=（湿重-干重）/湿重×100%。二、EB测定BBB通透性改变 利用尾静脉注射EB溶液，取损伤区脑组织检测EB含量，作为评价BBB通透性的指标，分析判定BBB通透性的变化趋势。三、AQP4mRNA原位杂交观察 利用原位杂交法鉴定AQP4mRNA在各实验处理组情况下损伤中心脑组织表达情况。并通过Image-proplus6.0图像分析软件计算全脑单位面积AQP4mRNA平均阳性强度。四、AQP4、ZO-1免疫荧光组织化学观察 利用免疫荧光组织化学检测在各实验处理组情况下AQP4的表达情况，分析判断TBI后AQP4的表达变化情况和脑水肿发生发展的相关性及鉴定干扰效果；检测ZO-1表达变化情况，分析和印证各实验组BBB通透性变化。五、光电镜观察细胞形态学变化及超微结构改变 各实验组大鼠分时间点，制作石蜡切片及超薄切片，分别用普通光学显微镜及透射电子显微镜进行观察。观测各实验处理后大鼠脑组织细胞形态学改变和BBB结构变化及超微结构改变。六、统计学分析 SPSS17.0版统计分析软件用于分析数据。根据数据的性质选取单因素方差分析或Pearson相关系数等分析方法。P值小于0.05视为差异有统计学意义。 结果 RNAi质粒可有效减少AQP4在受损脑组织的表达；RNAi组在各检测时间点脑组织含水量和BBB通透性均小于TBI组和对照质粒组(P0.05)；TBI后RNAi组ZO-1表达水平各时间点明显高于单纯创伤组和对照粒组（P＜0.05）；光镜下可见：RNAi组大鼠星形胶质细胞和神经元水肿的范围和程度均低于单纯创伤组和对照质粒组；假手术组胶质细胞和神经元未见明显的水肿。透射电镜下可见：RNAi组大鼠超微结构改变、紧密连接开放程度均低于单纯创伤组和对照质粒组；假手术组超微结构改变和紧密连接（tight junction，TJ）未见明显的改变。 结论 颅脑创伤后AQP4蛋白的表达变化趋势和脑水肿的发展趋势相一致，二者紧密相关；应用RNA干扰方法能有效减少AQP4的表达，可有效减少TBI后脑水肿BBB结构的破坏，对BBB起保护作用。
[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
本文编号：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
本文编号：2287761
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