本文選題：癲癇 + Rho激酶2　； 參考：《第四軍醫(yī)大學(xué)》2014年碩士論文

【摘要】：背景及目的: 癲癇是一種由于大腦神經(jīng)元放電異常所導(dǎo)致的短暫性中樞神經(jīng)系統(tǒng)功能失常為主要特征的慢性腦部疾病，癲癇的臨床表現(xiàn)主要有運(yùn)動(dòng)功能失調(diào)、意識(shí)障礙、感覺異常、精神紊亂、行為改變、自主神經(jīng)功能紊亂等。全世界約有0．5％至1％的人群受到該疾病的累及[1]。目前，在癲癇的動(dòng)物實(shí)驗(yàn)研究方面，可供選擇的動(dòng)物模型有很多種，然而氯化鋰-匹魯卡品癲癇大鼠模型是其中較為重要的一種。這種模型屬于癲癇持續(xù)狀態(tài)模型，這種模型與人類顳葉癲癇很多方面非常相似。例如：在發(fā)作表現(xiàn)、腦電圖改變、腦組織病理及藥物代謝等方面。所以，，一直以來被廣泛應(yīng)用于顳葉癲癇的研究工作中 @。 RhoA是一種普遍存在的小三磷酸鳥苷(Guanosine triphosphate,GTP)酶，是Ras超家族中的重要一員。包括腦組織在內(nèi)的各種組織及細(xì)胞中均可檢測(cè)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）兩種亞型。Rho激酶1主要在心臟、肺、骨骼肌等非神經(jīng)元組織表達(dá)。在神經(jīng)系統(tǒng)中主要表達(dá)的是ROCK2[4]。Rho/Rho激酶信號(hào)通路主要功能是參與調(diào)控各種免疫細(xì)胞、平滑肌細(xì)胞、炎性細(xì)胞等多種細(xì)胞的增殖與凋亡、基因轉(zhuǎn)錄、粘附與遷移及各種特異性功能的維持等生物學(xué)活動(dòng)，同時(shí)它還在多種心腦血管疾病的發(fā)生與發(fā)展過程中發(fā)揮重要作用。最初人們是在研究大腦皮層和海馬區(qū)的椎體神經(jīng)元細(xì)胞的過程中發(fā)現(xiàn)的ROCK2[5]。Rho激酶2參與多種神經(jīng)系統(tǒng)疾病的發(fā)病過程，并在這些疾病的發(fā)展過程中發(fā)揮重要作用，主要的疾病有：腦梗塞、短暫性腦缺血發(fā)作、阿爾茨海默爾病Alzheimer�。┮约凹顾钃p傷性疾病等。Rho激酶2的活化可以引起受損的神經(jīng)元軸突的回縮[6,7]。另外，在促進(jìn)神經(jīng)元細(xì)胞軸突的再生性生長(zhǎng)方面ROCK抑制劑也發(fā)揮著重要的作用[8]。法舒地爾是目前唯一應(yīng)用于臨床的ROCK抑制劑，主要作為鈣離子拮抗劑應(yīng)用在蛛網(wǎng)膜下腔出血所引起的腦血管痙攣的治療中[9]。 目前關(guān)于Rho激酶2在癲癇發(fā)病機(jī)制中作用的研究不是很多，但是二者的相互關(guān)系越來越受到大家的關(guān)注。在研究動(dòng)物癲癇模型和癲癇病人過程中，有學(xué)者發(fā)現(xiàn)的腦組織中神經(jīng)元樹突脊數(shù)量的減少，而ROCK2的一個(gè)主要作用就是在神經(jīng)元樹突的生長(zhǎng)過程中發(fā)揮著重要作用。目前有研究表明在神經(jīng)膠質(zhì)細(xì)胞表面興奮性氨基酸轉(zhuǎn)運(yùn)體（Excitatory amino acid transporter，EAAT）的表達(dá)過程中Rho激酶2發(fā)揮重要作用，并且EAAT的功能可以被Rho激酶抑制劑提高[10]。 N-甲基-D-天冬氨酸（NMDA）受體是在癲癇的發(fā)作過程中以及癲癇發(fā)作后的大腦繼發(fā)性損害中均發(fā)揮重要作用，被認(rèn)為是癲癇發(fā)病機(jī)制中重要的一個(gè)環(huán)節(jié)。雖然對(duì)于在神經(jīng)系統(tǒng)疾病中Rho/Rho激酶信號(hào)通路的作用人們已經(jīng)進(jìn)行了廣泛而深入的研究，但是關(guān)于ROCK在癲癇疾病的發(fā)生及發(fā)展過程中的作用的研究仍然很少被報(bào)道。最近的一篇文獻(xiàn)報(bào)道表明，在海人酸（Kainic acid，KA）模型的癲癇大鼠中Rho激酶2的表達(dá)增加，并且Rho激酶抑制劑Y-27632具有抗癲癇作用 @。目前還沒有關(guān)于氯化鋰-匹魯卡品癲癇大鼠腦內(nèi)Rho激酶2變化的研究，并且沒有關(guān)于Rho激酶抑制劑對(duì)癲癇大鼠腦電圖影響的研究。因此，本研究擬觀察氯化鋰-匹魯卡品癲癇大鼠海馬區(qū)Rho激酶2的表達(dá)情況，初步探討Rho/ROCK通路在癲癇疾病中的作用，并研究Rho激酶抑制劑對(duì)癲癇大鼠腦電圖的影響。 方法： 將健康雄性SD大鼠，隨機(jī)分入正常對(duì)照組、6h組、1d組、3d組、5d組、7d組、空白組、癲癇組和法舒地爾組。采用腹腔注射氯化鋰-匹魯卡品建立癲癇模型，按照Racine標(biāo)準(zhǔn)進(jìn)行觀察評(píng)分，通過免疫組織化學(xué)和Western blot的方法，比較6h組、1d組、3d組、5d組、7d組和對(duì)照組在大鼠顳葉、海馬區(qū)ROCK2的表達(dá)差異。對(duì)空白組、癲癇組及法舒地爾組大鼠進(jìn)行相應(yīng)的處理后，進(jìn)行腦電監(jiān)測(cè)，分析腦電圖變化。 結(jié)果： 實(shí)驗(yàn)一：Rho激酶2在癲癇大鼠海馬及顳葉腦組織表達(dá)的變化 （1）采用Western blot的方法檢測(cè)各組大鼠海馬組織中ROCK2表達(dá)量的變化，與對(duì)照組相比ROCK2的表達(dá)水平從癲癇發(fā)作后6h組開始逐漸升高，3d組達(dá)到最大值，5d、7d組又開始逐漸下降，3d組、5d組與對(duì)照組相比差異有統(tǒng)計(jì)學(xué)意義(P0.05)。采用Western blot的方法檢測(cè)各組大鼠顳葉腦組織中ROCK2表達(dá)量的變化，與對(duì)照組相比ROCK2的表達(dá)水平在癲癇發(fā)作后3d、5d、7d組均明顯升高，統(tǒng)計(jì)分析顯示差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 （2）采用免疫組化熒光雙標(biāo)的方法檢測(cè)ROCK2在大鼠海馬和顳葉腦組織中表達(dá)的部位，發(fā)現(xiàn)在熒光顯微鏡下正常組大鼠與實(shí)驗(yàn)組大鼠的海馬組織中ROCK2陽(yáng)性（綠色）與NeuN陽(yáng)性（紅色）在神經(jīng)元中共表達(dá)；顳葉腦組織中ROCK2陽(yáng)性（綠色）與GFAP陽(yáng)性（紅色）在星形膠質(zhì)細(xì)胞中共表達(dá)。 實(shí)驗(yàn)二：Rho激酶2抑制劑法舒地爾對(duì)癲癇大鼠腦電圖的影響 腦電監(jiān)測(cè)顯示空白組大鼠腦電正常，未見癲癇樣腦電波形。癲癇組大鼠在給予匹魯卡品約28min后出現(xiàn)癇樣放電波形，與癲癇組相比，法舒地爾組出現(xiàn)癇樣波形的潛伏時(shí)間明顯延長(zhǎng)，頻率明顯減慢、振幅明顯降低(P0.05)。 結(jié)論： （1）大鼠癲癇發(fā)作后急性期顳葉和海馬區(qū)腦組織中Rho激酶2的表達(dá)升高。 （2）在癲癇大鼠顳葉和海馬腦組織中，Rho激酶2在神經(jīng)元及膠質(zhì)細(xì)胞中均有表達(dá)。 （3） Rho激酶2抑制劑法舒地爾對(duì)于癲癇模型大鼠癲癇的發(fā)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 .
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R742.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 姚君茹,潘三強(qiáng),呂來清,高秀來;慢性癲癇模型大鼠腦谷氨酸神經(jīng)元的變化[J];解剖科學(xué)進(jìn)展;2004年01期

2 潘映輻;癲癇診斷中病史和腦電圖的臨床意義[J];中國(guó)醫(yī)刊;2002年04期

本文編號(hào)：1975192