本文選題：癲癇 + Nrf2��； 參考：《河北醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：癲癇是多種病因引起的慢性腦部疾病,通過引起大腦神經(jīng)元反復(fù)地、過度地超同步化放電，導(dǎo)致中樞神經(jīng)系統(tǒng)功能短暫性失常的臨床表現(xiàn)。近年的研究結(jié)果表明,人體內(nèi)過多的自由基(Free Radical, FR)積累與許多慢性疾病都與有關(guān)。氧自由基導(dǎo)致的氧化應(yīng)激損傷目前已被廣泛研究證實為癲癇發(fā)病的重要機制之一。已經(jīng)有研究證明，無論是癲癇動物實驗?zāi)Ｐ瓦�是癲癇患者的腦內(nèi)均存在著活躍的自由基反應(yīng)，癲癇發(fā)作時自由基進一步明顯增加，遠遠超過機體對自由基的清除能力，導(dǎo)致神經(jīng)元和線粒體的損傷,最終可加重癲癇的發(fā)生。清除氧自由基、抗氧化的治療方法能為癲癇的藥物治療提供新的靶點。 轉(zhuǎn)錄因子NF-E2相關(guān)因子(NF-E2-related factor2，Nrf2)是細胞氧化應(yīng)激反應(yīng)中的關(guān)鍵因子，Nrf2通過與抗氧化反應(yīng)元件（antioxidant responseelement，ARE）相互作用來調(diào)節(jié)抗氧化蛋白和Ⅱ相解毒酶的表達。既往研究證實，Nrf2-ARE通路對神經(jīng)系統(tǒng)疾病如：肌萎縮側(cè)索硬化癥(ALS)、帕金森病、腦出血、腦梗死和腦外傷等，具有很強的神經(jīng)保護功能，但是其在癲癇發(fā)病中抗氧化應(yīng)激損傷的相關(guān)報道甚少，本實驗主要研究戊四氮（pentylenetetrazoloe，PTZ）點燃慢性癲癇小鼠腦內(nèi)Nrf2-ARE通路的保護作用。使用基因型為Nrf2(+)和基因型為Nrf2(-)的兩種小鼠制作戊四氮（pentylenetetrazoloe，PTZ）點燃慢性癲癇小鼠模型，觀察Nrf2-ARE通路阻斷后戊四氮致癇小鼠癇性發(fā)作等級、小鼠海馬CA1區(qū)神經(jīng)細胞損傷情況及海馬組織中Nrf-ARE通路表達產(chǎn)物血紅素氧合酶-1(heme oxygenase1，HO-1)表達量的變化情況，評價Nrf2-ARE通路對癲癇過程中神經(jīng)細胞的保護作用并對其機制進行初步探討，為闡明癲癇的病理生理機制提供實驗依據(jù)，同時為癲癇治療提供新的靶點，為抗癲癇藥物的選擇提供新的思路。 方法：選擇實驗動物并分組：選擇健康成年雄性C57/B小鼠，基因型為Nrf2(+)、經(jīng)PCR鑒定基因型為（Nrf2-/-）的成年雄性Nrf2基因敲除（Nrf2-KO）小鼠，由河北醫(yī)科大學(xué)動物實驗中心及省二院神經(jīng)內(nèi)科實驗室提供，適應(yīng)性飼養(yǎng)1周后，隨機分成四組：C57小鼠對照組（C-Control）n=30、C57小鼠致癇組（C-PTZ）n=30、Nrf2基因敲除小鼠對照組（N-Control）n=30、Nrf2基因敲除小鼠致癇組（N-PTZ）n=30。 模型的制備：癲癇組采用戊四氮慢速點燃癲癇模型：腹腔注射給藥：隔天給藥，26天內(nèi)給藥13次，每次均為上午9:00-11:30進行。給藥前稱體重，致癇組每只小鼠腹腔注射0.5%的PTZ，劑量為32mg/kg，，合0.0064ml/g，對照組按照0.0064ml/g給予生理鹽水腹腔注射。注射后將小鼠放至透明箱中觀察30min，記錄小鼠癲癇發(fā)作的最大級別。癇性發(fā)作的發(fā)作等級變化按照Racine六級評價標準：0級，無任何反應(yīng)；Ⅰ級，濕狗樣抖動、面部抽搐及咀嚼；Ⅱ級，頸部肌肉痙攣表現(xiàn)為點頭和/或甩尾；Ⅲ級，一側(cè)前肢陣攣；Ⅳ級，雙側(cè)前肢陣攣伴立；Ⅴ級，全身陣攣，失去平衡，跌倒。戊四氮點燃過程：采用慢點燃法，隔天給予小鼠PTZ。連續(xù)三次記錄到4~5級點燃即為點燃成功，納入實驗。造模成功后繼續(xù)隔日給予戊四氮刺激一次，誘導(dǎo)癲癇發(fā)作。制模過程中小鼠出現(xiàn)死亡或不符合模型要求者，予以剔除并隨機補充，保證每組數(shù)量30只。 取材：最后一次PTZ刺激結(jié)束48小時后，每組隨機選取5只小鼠用于尼氏染色技術(shù)，麻醉后用4%的多聚甲醛液灌流固定后，取小鼠海馬CA1區(qū)域的腦組織標本制作成蠟塊，進行尼氏染色，測定小鼠海馬CA1區(qū)神經(jīng)細胞數(shù)；每組隨機選取5只小鼠麻醉后用1%多聚甲醛-1.25%戊二醛0.1M磷酸緩沖液灌流固定后，取小鼠海馬區(qū)域的腦組織標本，經(jīng)透射電子顯微鏡觀察海馬組織CA1區(qū)神經(jīng)元亞細胞結(jié)構(gòu)；每組取20只小鼠麻醉后快速在冰塊上提取小鼠海馬組織，利用western blot技術(shù)半定量測定HO-1的含量。 結(jié)果： 1四組小鼠在癇性發(fā)作等級方面的比較：N-PTZ組比C-PTZ組的平均癇性發(fā)作等級顯著升高（P0.05）；N-Control組與C-Control組均無癇性發(fā)作。 2四組小鼠腦組織尼氏染色海馬CA1區(qū)神經(jīng)元細胞數(shù)目的比較：致癇組均比空白對照組的神經(jīng)元數(shù)目明顯減少（P0.05）；N-PTZ組比C-PTZ組的神經(jīng)元數(shù)目顯著減少（P0.05）；N-Control組與C-Control組差異無統(tǒng)計學(xué)意義（P0.05）。 3四組小鼠腦組織海馬神經(jīng)元透射電鏡觀察結(jié)果比較：電鏡觀察示，空白對照組，即C-control組及N-control組：海馬CA1區(qū)細胞大體結(jié)構(gòu)未發(fā)現(xiàn)異常。細胞內(nèi)神經(jīng)元核膜清晰，胞質(zhì)中細胞器豐富，胞質(zhì)內(nèi)可見線粒體、粗面內(nèi)質(zhì)網(wǎng)和大量的多核糖體。N-PTZ組：海馬CA1區(qū)神經(jīng)元細胞可見明顯的大體結(jié)構(gòu)損傷，出現(xiàn)細胞體的破碎。細胞內(nèi)核膜內(nèi)陷或邊界不清，核質(zhì)不均勻，異染色質(zhì)積聚成塊、增多、邊集，細胞質(zhì)中線粒體出現(xiàn)腫脹、線粒體嵴發(fā)生斷裂甚至空化，粗面內(nèi)質(zhì)網(wǎng)池擴張、附著的核糖體脫落，游離核糖體解聚，重者出現(xiàn)胞漿空化。C-PTZ組：海馬CA1區(qū)有部分神經(jīng)元受損，且受損程度較N-PTZ組明顯較輕，表現(xiàn)為核膜邊界不清，細胞變形，電子密度影呈水腫表現(xiàn)，少數(shù)細胞質(zhì)濃縮，粗面內(nèi)質(zhì)網(wǎng)排列紊亂，多聚核糖體稍減少，部分線粒體有嵴斷裂現(xiàn)象。 4四組小鼠海馬區(qū)腦組織HO-1的表達western blot半定量比較：N-control組比C-control組表達量減少（P0.05）；N-PTZ組比C-PTZ組HO-1表達量明顯減少（P0.05）；N-PTZ組比N-control組HO-1表達量明顯增多（P0.05）；C-PTZ組比C-control組HO-1表達量明顯增多（P0.05）。 結(jié)論： 戊四氮點燃的慢性癲癇模型能夠很好模擬了人類海馬神經(jīng)元的損傷。Nrf2基因缺陷小鼠的平均癲癇發(fā)作等級更高，且其海馬區(qū)神經(jīng)元在癲癇病程中受到更大的損傷，與此同時海馬組織中血紅素氧合酶-1的含量明顯降低。這可能表示癲癇癲癇病程中，Nrf2基因可能對癲癇小鼠的海馬神經(jīng)元有保護作用，且可能是通過激活Nrf2-ARE通路進而增加腦組織中HO-1的表達來實現(xiàn)的。
[Abstract]:Objective: epilepsy is a chronic brain disease caused by a variety of causes, which results in a clinical manifestation of transient dysfunction of the central nervous system by overly oversynchronous discharge caused by the recurrent brain neurons. Recent results show that the accumulation of Free Radical (FR) in the human body is associated with many chronic diseases. Oxidative stress damage caused by oxygen free radicals has been widely studied as one of the important mechanisms of epilepsy. Research has shown that there is an active free radical reaction in both epileptic animal model and epileptic patients, and the free radicals in epileptic seizures are further increased, far more than the body's freedom. The scavenging ability of the base leads to the damage of neurons and mitochondria, which can eventually aggravate the occurrence of epilepsy. Scavenging oxygen free radicals and antioxidation therapy can provide new targets for the treatment of epilepsy.
The transcription factor NF-E2 related factor (NF-E2-related FACTOR2, Nrf2) is a key factor in cellular oxidative stress. Nrf2 regulates the expression of antioxidant protein and phase II detoxification enzyme by interacting with the antioxidant response element (antioxidant responseelement, ARE). Previous studies have confirmed that the Nrf2-ARE pathway is a disease of the nervous system, such as the muscle. Amyotrophic lateral sclerosis (ALS), Parkinson's disease, cerebral hemorrhage, cerebral infarction and brain injury have strong neuroprotective functions. However, there are few reports on the damage of antioxidant stress in epilepsy. This experiment mainly studies the protective effect of pentylenetetrazoloe (PTZ) on the protection of Nrf2-ARE pathway in the brain of chronic epileptic mice. Two mice with genotype Nrf2 (+) and genotype Nrf2 (-) were used to make amyl four nitrogen (pentylenetetrazoloe, PTZ) to kindle chronic epilepsy in mice. The level of epileptic seizures in epileptic mice induced by Nrf2-ARE pathway, the damage of neurons in CA1 region of hippocampus and the heme oxygenation of the expression products of Nrf-ARE pathway in hippocampus of hippocampus were observed. The change of the expression of enzyme -1 (heme oxygenase1, HO-1), to evaluate the protective effect of Nrf2-ARE pathway to the neural cells in the process of epilepsy and to discuss its mechanism, provide experimental basis for elucidating the pathophysiological mechanism of epilepsy, and provide new targets for the treatment of epilepsy, and provide a new idea for the selection of antiepileptic drugs.
Methods: select experimental animals and group: select healthy adult male C57/B mice and genotype Nrf2 (+). The adult male Nrf2 gene knockout (Nrf2-KO) mice were identified by PCR (Nrf2-/-), which were provided by the animal experiment center of Hebei Medical University and the second hospital of the province. After 1 weeks of adaptive feeding, they were randomly divided into four groups: C 57 mice control group (C-Control) n=30, C57 mice induced epilepsy group (C-PTZ) n=30, Nrf2 gene knockout mice control group (N-Control) n=30, Nrf2 knockout mice induced seizure group (N-PTZ) n=30.
Model preparation: epileptic group was treated with an amyl four nitrogen slow kindling model of epilepsy: intraperitoneal injection: medication every other day and 13 times in 26 days, each time was 9:00-11:30 a.m., before the drug was given weight, each mice in the epilepsy group were injected with 0.5% PTZ, the dose was 32mg/kg, and 0.0064ml/g, and the control group was given the saline abdomen according to 0.0064ml/g. The mice were injected into the cavity. The mice were placed in the transparent box to observe 30min and record the maximum level of epileptic seizures in mice. The level of seizures was changed according to the standard of Racine six: grade 0, without any reaction; grade I, wet dog jitter, facial convulsion and chewing; class II, neck muscle spasms were nodding and / or tail flick; class III, One side of the forelimb clonus; grade IV, bilateral forelimb clonus and clonus; grade V, general clonus, loss of balance, fall. The amyl four nitrogen kindling process: using the slow ignition method, the mice were given three consecutive records to the PTZ. kindling every other day to kindle successfully, which was brought into the experiment. After the success of the model, the stimulation of the amyl four nitrogen was given and the epileptic seizures were induced. The mice died during the mold making process or did not meet the requirements of the model. They were eliminated and randomly supplemented to ensure that there were 30 in each group.
48 hours after the final PTZ stimulation, 5 mice in each group were randomly selected for Nissl staining. After anesthesia, the brain tissue samples from the CA1 region of the hippocampus of the mice were made into wax blocks, and Nissl staining was performed to determine the number of neurons in the CA1 region of the hippocampus of mice. 5 mice were randomly selected for each group. After drunken injection of 1% polyformaldehyde -1.25% glutaraldehyde 0.1M phosphate buffer, the brain tissue specimens of the hippocampus region of mice were collected, and the subcellular structure of neurons in the hippocampal CA1 region was observed by transmission electron microscope. 20 mice in each group were extracted and extracted quickly on the ice block. Semi quantitative determination by Western blot technique was used. The content of HO-1.
Result錛

本文編號：1786027