Rho激酶2在癫痫大鼠脑内表达变化及其机制的研究

发布时间：2018-06-04 01:25

本文选题：癫痫 + Rho激酶2　；参考：《第四军医大学》2014年硕士论文

【摘要】：背景及目的: 癫痫是一种由于大脑神经元放电异常所导致的短暂性中枢神经系统功能失常为主要特征的慢性脑部疾病，癫痫的临床表现主要有运动功能失调、意识障碍、感觉异常、精神紊乱、行为改变、自主神经功能紊乱等。全世界约有0．5％至1％的人群受到该疾病的累及[1]。目前，在癫痫的动物实验研究方面，可供选择的动物模型有很多种，然而氯化锂-匹鲁卡品癫痫大鼠模型是其中较为重要的一种。这种模型属于癫痫持续状态模型，这种模型与人类颞叶癫痫很多方面非常相似。例如：在发作表现、脑电图改变、脑组织病理及药物代谢等方面。所以，，一直以来被广泛应用于颞叶癫痫的研究工作中 @。 RhoA是一种普遍存在的小三磷酸鸟苷(Guanosine triphosphate,GTP)酶，是Ras超家族中的重要一员。包括脑组织在内的各种组织及细胞中均可检测到RhoA[3]。Rho激酶（Rho-associated protein kinase，ROCK）是RhoA的重要下游分子，包括Rho激酶1（Rho-associated protein kinase1，ROCK1）和Rho激酶2（Rho-associated proteinkinase2，ROCK2）两种亚型。Rho激酶1主要在心脏、肺、骨骼肌等非神经元组织表达。在神经系统中主要表达的是ROCK2[4]。Rho/Rho激酶信号通路主要功能是参与调控各种免疫细胞、平滑肌细胞、炎性细胞等多种细胞的增殖与凋亡、基因转录、粘附与迁移及各种特异性功能的维持等生物学活动，同时它还在多种心脑血管疾病的发生与发展过程中发挥重要作用。最初人们是在研究大脑皮层和海马区的椎体神经元细胞的过程中发现的ROCK2[5]。Rho激酶2参与多种神经系统疾病的发病过程，并在这些疾病的发展过程中发挥重要作用，主要的疾病有：脑梗塞、短暂性脑缺血发作、阿尔茨海默尔病Alzheimer病）以及脊髓损伤性疾病等。Rho激酶2的活化可以引起受损的神经元轴突的回缩[6,7]。另外，在促进神经元细胞轴突的再生性生长方面ROCK抑制剂也发挥着重要的作用[8]。法舒地尔是目前唯一应用于临床的ROCK抑制剂，主要作为钙离子拮抗剂应用在蛛网膜下腔出血所引起的脑血管痉挛的治疗中[9]。目前关于Rho激酶2在癫痫发病机制中作用的研究不是很多，但是二者的相互关系越来越受到大家的关注。在研究动物癫痫模型和癫痫病人过程中，有学者发现的脑组织中神经元树突脊数量的减少，而ROCK2的一个主要作用就是在神经元树突的生长过程中发挥着重要作用。目前有研究表明在神经胶质细胞表面兴奋性氨基酸转运体（Excitatory amino acid transporter，EAAT）的表达过程中Rho激酶2发挥重要作用，并且EAAT的功能可以被Rho激酶抑制剂提高[10]。 N-甲基-D-天冬氨酸（NMDA）受体是在癫痫的发作过程中以及癫痫发作后的大脑继发性损害中均发挥重要作用，被认为是癫痫发病机制中重要的一个环节。虽然对于在神经系统疾病中Rho/Rho激酶信号通路的作用人们已经进行了广泛而深入的研究，但是关于ROCK在癫痫疾病的发生及发展过程中的作用的研究仍然很少被报道。最近的一篇文献报道表明，在海人酸（Kainic acid，KA）模型的癫痫大鼠中Rho激酶2的表达增加，并且Rho激酶抑制剂Y-27632具有抗癫痫作用 @。目前还没有关于氯化锂-匹鲁卡品癫痫大鼠脑内Rho激酶2变化的研究，并且没有关于Rho激酶抑制剂对癫痫大鼠脑电图影响的研究。因此，本研究拟观察氯化锂-匹鲁卡品癫痫大鼠海马区Rho激酶2的表达情况，初步探讨Rho/ROCK通路在癫痫疾病中的作用，并研究Rho激酶抑制剂对癫痫大鼠脑电图的影响。方法：将健康雄性SD大鼠，随机分入正常对照组、6h组、1d组、3d组、5d组、7d组、空白组、癫痫组和法舒地尔组。采用腹腔注射氯化锂-匹鲁卡品建立癫痫模型，按照Racine标准进行观察评分，通过免疫组织化学和Western blot的方法，比较6h组、1d组、3d组、5d组、7d组和对照组在大鼠颞叶、海马区ROCK2的表达差异。对空白组、癫痫组及法舒地尔组大鼠进行相应的处理后，进行脑电监测，分析脑电图变化。结果：实验一：Rho激酶2在癫痫大鼠海马及颞叶脑组织表达的变化（1）采用Western blot的方法检测各组大鼠海马组织中ROCK2表达量的变化，与对照组相比ROCK2的表达水平从癫痫发作后6h组开始逐渐升高，3d组达到最大值，5d、7d组又开始逐渐下降，3d组、5d组与对照组相比差异有统计学意义(P0.05)。采用Western blot的方法检测各组大鼠颞叶脑组织中ROCK2表达量的变化，与对照组相比ROCK2的表达水平在癫痫发作后3d、5d、7d组均明显升高，统计分析显示差异有统计学意义(P0.05)。（2）采用免疫组化荧光双标的方法检测ROCK2在大鼠海马和颞叶脑组织中表达的部位，发现在荧光显微镜下正常组大鼠与实验组大鼠的海马组织中ROCK2阳性（绿色）与NeuN阳性（红色）在神经元中共表达；颞叶脑组织中ROCK2阳性（绿色）与GFAP阳性（红色）在星形胶质细胞中共表达。实验二：Rho激酶2抑制剂法舒地尔对癫痫大鼠脑电图的影响脑电监测显示空白组大鼠脑电正常，未见癫痫样脑电波形。癫痫组大鼠在给予匹鲁卡品约28min后出现痫样放电波形，与癫痫组相比，法舒地尔组出现痫样波形的潜伏时间明显延长，频率明显减慢、振幅明显降低(P0.05)。结论：（1）大鼠癫痫发作后急性期颞叶和海马区脑组织中Rho激酶2的表达升高。（2）在癫痫大鼠颞叶和海马脑组织中，Rho激酶2在神经元及胶质细胞中均有表达。（3） Rho激酶2抑制剂法舒地尔对于癫痫模型大鼠癫痫的发作具有一定的抑制作用。
[Abstract]:Background and Purpose :

Epilepsy is a kind of chronic brain disease characterized by transient central nervous system dysfunction caused by abnormal discharge of cerebral nerve . The clinical manifestations of epilepsy mainly include motor dysfunction , consciousness disorder , sensory abnormality , mental disorder , behavior change , autonomic nervous function disorder , etc . Approximately 0.5 % to 1 % of the population in the world are affected by the disease . At present , there are many animal models of epilepsy in the field of epilepsy . However , the model of epileptic rats with lithium chloride - pilocarpine is one of the most important models . This model belongs to the model of the status epilepticus . This model is very similar to many aspects of human temporal lobe epilepsy . For example , it has been widely used in the research of temporal lobe epilepsy .

RhoA is a ubiquitous guanosine triphosphate ( GTP ) enzyme , which is an important member of the Ras superfamily . RhoA can be detected in various tissues and cells including brain tissue . rho - associated protein kinase ( ROCK ) is an important downstream molecule of RhoA , including the two subtypes of rho kinase 1 ( rho - associated protein kinase1 , rock 1 ) and rho - associated protein kinase2 , ( 2 ) . The main expression of rho kinase 1 in heart , lung , skeletal muscle and other non - neuronal tissues . The main function of the signal pathway is to regulate the proliferation and apoptosis of various kinds of cells such as immune cells , smooth muscle cells , inflammatory cells and so on . It also plays an important role in the development and development of various cardiovascular and cerebrovascular diseases . It plays an important role in the development of these diseases , including cerebral infarction , transient ischemic attack , Alzheimer ' s disease , and spinal cord injury . fasudil is the only ROCK inhibitor currently used in clinic , mainly used as calcium ion antagonist in the treatment of cerebrovascular spasm caused by subarachnoid hemorrhage .

In the study of animal epilepsy model and epilepsy , the number of dendritic spines in the brain tissue is decreased , and one of the main functions of the two is the important role in the growth of neuronal dendritic cells . N - methyl - D - aspartate ( NMDA ) receptor plays an important role in the seizure of epilepsy and secondary damage to the brain after seizures .

Method :

SD rats were randomly divided into normal control group , 6h group , 1d group , 3d group , 5d group , 7d group , blank group , epilepsy group and fasudil group .

Results :

Experiment 1 : Changes of expression of rho kinase 2 in hippocampus and temporal lobe of epileptic rats

( 1 ) Western blot was used to detect the change of the expression level in the hippocampus of each group . Compared with the control group , the expression level increased gradually from 6 hours after the seizure , and the difference was statistically significant in the 3d group and the 5d group compared with the control group ( P0.05 ) .

( 2 ) In the hippocampus and temporal lobe brain tissues of rats by immunohistochemical method , the expression of 2 positive ( green ) and NeuN positive ( red ) were detected in hippocampus and temporal lobe of rats .
2 positive ( green ) and GFAP - positive ( red ) in the brain tissue of temporal lobe were co - expressed in astrocytes .

Experiment 2 : Effect of Shudil on Electroencephalogram in Rats with Epilepsy

EEG monitoring showed that the EEG was normal in the blank group , and no epileptic brain was found in the epileptic group . The seizure - like discharge waveform occurred in the epileptic group after about 28 min . The latency of the epileptic wave appeared to be significantly prolonged and the amplitude decreased significantly compared with the epilepsy group ( P0.05 ) .

Conclusion :

( 1 ) The expression of rho kinase 2 in the brain tissue of the temporal lobe and hippocampus was increased in the acute stage of rats after seizure .

( 2 ) In the temporal lobe of epileptic rats and the brain tissue of hippocampus , the rho kinase 2 was expressed in neurons and glial cells .

( 3 ) Shudil kinase 2 inhibitor fasudil has a certain inhibitory effect on the seizure of epileptic model rats .
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.1

【参考文献】