【摘要】：顳葉內(nèi)側(cè)癲癇(Mesial temporal lobe epilepsy,MTLE)是成人常見的局灶性癲癇。75%左右的MTLE患者因?qū)ΜF(xiàn)有的抗癲癇藥物不敏感而最終發(fā)展成為藥物難治性癲癇,目前臨床上常通過外科手術(shù)切除致癇病灶來控制其癲癇發(fā)作。盡管人們在不斷的探索和研究MTLE的發(fā)病機制,但截止目前,其具體的發(fā)生機制仍不清楚。新近研究發(fā)現(xiàn),一方面,反復的癲癇發(fā)作常引起大腦產(chǎn)生神經(jīng)炎癥反應,并伴有血腦屏障的功能損傷。另一方面,神經(jīng)炎癥反應和血腦屏障功能損傷又可促進MTLE的發(fā)生發(fā)展。這二者之間以正反饋機制形成一個惡性循環(huán),并以此促進癲癇的發(fā)生發(fā)展。來自MTLE臨床標本和動物模型的證據(jù)顯示,血管內(nèi)皮生長因子(vascular endothelial growth factor,VEGF)在調(diào)控血腦屏障通透性及神經(jīng)炎癥方面具有重要作用。VEGF家族包括7個成員,分別是VEGF,VEGF-B,VEGF-C,VEGF-D,VEGF-E,VEGF-F,以及胎盤生長因子。既往的研究已經(jīng)對VEGF在多種神經(jīng)系統(tǒng)疾病中的作用做了較為全面和深入的研究,包括MTLE。除VEGF外,近年來越來越多的研究開始關注VEGF-C在中樞神經(jīng)系統(tǒng)中的作用。VEGF-C是一種重要的調(diào)控淋巴管及血管生成的長生因子,通過與其受體VEGFR-2和VEGFR-3結(jié)合而發(fā)揮相應的生物學作用。已有研究表明,VEGF-C與神經(jīng)炎癥及血腦屏障通透性關系密切,例如VEGF-C高表達于缺血性腦卒中及骨髓移植后大鼠大腦的反應性星形膠質(zhì)細胞和小膠質(zhì)細胞上;VEGF-C及其受體VEGFR-2和VEGFR-3還高表達于與兒童難治性癲癇密切相關的結(jié)節(jié)性(Tuberous sclerosis complex,TSC)硬化皮層結(jié)節(jié)的反應性星形膠質(zhì)細胞上;此外,在大鼠的帕金森模型中,VEGF-C可明顯增加其血腦屏障的通透性。那么,VEGF-C在MTLE的表達模式是怎樣的?其在MTLE發(fā)生中的作用及機制又是什么?這些都是值得我們關注和進一步研究的問題。因此,在本研究中,我們首先通過實時定量PCR,Western blot和免疫組化等技術(shù)觀察VEGF-C及其受體在MTLE患者手術(shù)切除的病灶中的表達和細胞定位;然后在匹羅卡品誘導的小鼠MTLE模型中使用Western blot,視頻腦電檢分別測VEGF-C及其受體在MTLE小鼠癲癇發(fā)作的急性期(3小時),潛伏期(1周)和慢性自發(fā)性癲癇發(fā)作期(3周)的表達和對癲癇發(fā)作的影響;同時使用免疫酶聯(lián)反應(Enzyme-linked immune response,ELISA)檢測了VEGF-C及其受體在上述三個時間點對MTLE小鼠海馬中血腦屏障開放的標志分子白蛋白(Albumin),白介素1β(IL-1β)IL-6,IL-17和腫瘤壞死因子α(TNF-α)四種和MTLE發(fā)生發(fā)展密切相關的炎癥因子,以及mTOR信號通路(mTOR的激活以p-S6表達增加為標志)的作用。此外,由于在MTLE動物模型中我們發(fā)現(xiàn)VEGF-C可上調(diào)上述致炎因子,加之我們前期研究發(fā)現(xiàn)IL-17在與癲癇發(fā)作高度相關的大腦發(fā)育障礙中具有重要作用,所以我們還研究了IL-17及其受體IL-17R在MTLE患者手術(shù)切除的病灶中的表達和分布。通過上述研究,我們得到如下結(jié)果:1.VEGF-C,VEGFR-2和VEGFR-3在MTLE患者致癇灶及腦脊液和血清中的表達水平較對照組明顯增高。2.免疫組化及熒光雙標結(jié)果顯示,VEGF-C高表達于MTLE病灶的神經(jīng)元,反應性星形膠質(zhì)細胞,以及血管內(nèi)皮細胞上,VEGFR-2高表達于反應性星形膠質(zhì)細胞和血管內(nèi)皮細胞上,而VEGFR3則僅高表達于反應性星形膠質(zhì)細胞上。3.在匹羅卡品誘導的小鼠MTLE模型中,VEGF-C,VEGFR-2和VEGFR-3在癲癇發(fā)作的3 h,1w,和3 w的蛋白表達均顯著增高。4.視頻腦電監(jiān)測結(jié)果顯示,VEGF-C可明顯增加MTLE小鼠在上述三個時間點的棘波放電頻率和癲癇發(fā)作次數(shù),且該效應可被VEGFR-2或者VEGFR-3拮抗劑所阻斷。5.VEGF-C上調(diào)MTLE小鼠海馬在上述一個時間點白蛋白、IL-1β、IL-6、IL-17、TNF-α、p-S6的表達,VEGFR-2拮抗劑可阻斷上述效應,VEGFR3拮抗劑可阻斷除白蛋白之外的效應。6.IL-17和IL17R在MTLE患者致癇灶中的mRNA和蛋白表達水平較對照組明顯增高。7.IL-17和IL-17R高表達于MTLE病灶的神經(jīng)元,反應性星形膠質(zhì)細胞和小膠質(zhì)細胞,以及血管內(nèi)皮細胞上。我們的研究結(jié)果提示,VEGF-C信號通路在MTLE的發(fā)生中具有重要作用。
[Abstract]:Mesial temporal lobe epilepsy (MTLE) is a common focal epilepsy in adults, and the MTLE patients with localized epilepsy around.75% have developed into drug refractory epilepsy due to their insensitivity to existing antiepileptic drugs. At present, surgical excision of epileptic foci is often used to control epileptic seizures. So far, the pathogenesis of MTLE has been studied, but its specific mechanism is still unclear. Recently, recent studies have found that recurrent seizures often cause neuroinflammatory reactions in the brain, accompanied by functional damage to the blood brain barrier. On the other hand, the neuroinflammatory response and damage to the blood brain barrier can also promote the occurrence of MTLE. A positive feedback mechanism between the two forms a vicious cycle and promotes the development of epilepsy. Evidence from the MTLE clinical specimens and animal models shows that vascular endothelial growth factor (VEGF) plays an important role in regulating the permeability of the blood brain barrier and the neuroinflammation of the.VEGF family. It includes 7 members, VEGF, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and placental growth factors. Previous studies have done a more comprehensive and in-depth study of the role of VEGF in a variety of nervous system diseases, including MTLE. except VEGF. In recent years, more and more studies have begun to focus on the role of VEGF-C in the central nervous system. VEGF-C is an important growth factor that regulates lymphatic vessels and angiogenesis and plays a biological role by combining its receptor VEGFR-2 and VEGFR-3. Studies have shown that VEGF-C is closely related to neuroinflammation and blood brain barrier permeability. For example, VEGF-C is highly expressed in the brain of rats with hemorrhagic stroke and bone marrow transplantation. On reactive astrocytes and microglia, VEGF-C and its receptor VEGFR-2 and VEGFR-3 are also highly expressed in reactive astrocytes of the nodular (Tuberous sclerosis complex, TSC) hardened cortical nodules closely related to intractable epilepsy in children. In addition, VEGF-C can significantly increase the blood brain in the Parkinson model of rats. The permeability of the barrier. Then, what is the expression pattern of VEGF-C in the MTLE? What is its role and mechanism in the occurrence of MTLE? These are all issues worthy of our attention and further study. Therefore, in this study, we first observed VEGF-C and its receptor in MTLE by real-time quantitative PCR, Western blot and immunohistochemistry. The expression and cell location in the patient's excised lesion were then used in the pilocarpine induced mouse MTLE model with Western blot, and the video EEG was used to detect VEGF-C and its receptors in the acute phase of the epileptic seizures (3 hours) in MTLE mice, the latent period (1 weeks) and the expression of the chronic self epileptic seizures (3 weeks) and the shadow of the epileptic seizures. At the same time, Enzyme-linked immune response (ELISA) was used to detect the molecular albumin (Albumin) of VEGF-C and its receptor on the blood brain barrier in the hippocampus of MTLE mice at these three time points, and four kinds of interleukins 1 beta (IL-1 beta) IL-6, IL-17 and tumor necrosis factor alpha (TNF- alpha) were closely related to the development of MTLE. Inflammatory factors, as well as the role of the mTOR signaling pathway (the activation of the mTOR as a marker of p-S6 expression). In addition, as we found that VEGF-C can up-regulate the afore-mentioned inflammatory factors in the MTLE animal model, and our previous study found that IL-17 plays an important role in the development of brain disorders associated with epileptic seizures, so we also studied The expression and distribution of IL-17 and its receptor IL-17R in the surgical excised lesions of MTLE patients. Through these studies, we have obtained the following results: the expression level of 1.VEGF-C, VEGFR-2 and VEGFR-3 in the epileptic foci and cerebrospinal fluid and serum in MTLE patients is significantly higher than that in the control group. The results of.2. immunization and fluorescence double labeling show that VEGF-C is highly expressed in M. The neurons of TLE, reactive astrocytes, and vascular endothelial cells, VEGFR-2 were highly expressed on reactive astrocytes and vascular endothelial cells, while VEGFR3 was only highly expressed on reactive astrocytes of.3. in pilocarpine induced MTLE mold, and VEGF-C, VEGFR-2 and VEGFR-3 were 3 h in epileptic seizures. The protein expression of 1W, and 3 W increased significantly in.4. video EEG monitoring results, and the results showed that VEGF-C significantly increased the frequency of spike wave discharge and the number of epileptic seizures at the three time points of MTLE mice, and the effect could be increased by VEGFR-2 or VEGFR-3 antagonists on the up regulation of MTLE rat hippocampus at the same time point albumin, IL-1 beta. The expression of IL-6, IL-17, TNF- a, p-S6, VEGFR-2 antagonists block the above effects. VEGFR3 antagonists can block the effect of.6.IL-17 and IL17R in.6.IL-17 and IL17R in the epileptic foci of MTLE patients. The expression level of mRNA and protein is significantly higher than that of the control group, and the.7.IL-17 and IL-17R are higher in the neurons of the foci, reactive astrocytes and small Our findings suggest that VEGF-C signaling pathway plays an important role in the pathogenesis of MTLE.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R742.1

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