血管内皮生长因子-C在颞叶内侧癫痫中的作用及机制研究

发布时间：2018-07-29 13:08

【摘要】：颞叶内侧癫痫(Mesial temporal lobe epilepsy,MTLE)是成人常见的局灶性癫痫。75%左右的MTLE患者因对现有的抗癫痫药物不敏感而最终发展成为药物难治性癫痫,目前临床上常通过外科手术切除致痫病灶来控制其癫痫发作。尽管人们在不断的探索和研究MTLE的发病机制,但截止目前,其具体的发生机制仍不清楚。新近研究发现,一方面,反复的癫痫发作常引起大脑产生神经炎症反应,并伴有血脑屏障的功能损伤。另一方面,神经炎症反应和血脑屏障功能损伤又可促进MTLE的发生发展。这二者之间以正反馈机制形成一个恶性循环,并以此促进癫痫的发生发展。来自MTLE临床标本和动物模型的证据显示,血管内皮生长因子(vascular endothelial growth factor,VEGF)在调控血脑屏障通透性及神经炎症方面具有重要作用。VEGF家族包括7个成员,分别是VEGF,VEGF-B,VEGF-C,VEGF-D,VEGF-E,VEGF-F,以及胎盘生长因子。既往的研究已经对VEGF在多种神经系统疾病中的作用做了较为全面和深入的研究,包括MTLE。除VEGF外,近年来越来越多的研究开始关注VEGF-C在中枢神经系统中的作用。VEGF-C是一种重要的调控淋巴管及血管生成的长生因子,通过与其受体VEGFR-2和VEGFR-3结合而发挥相应的生物学作用。已有研究表明,VEGF-C与神经炎症及血脑屏障通透性关系密切,例如VEGF-C高表达于缺血性脑卒中及骨髓移植后大鼠大脑的反应性星形胶质细胞和小胶质细胞上;VEGF-C及其受体VEGFR-2和VEGFR-3还高表达于与儿童难治性癫痫密切相关的结节性(Tuberous sclerosis complex,TSC)硬化皮层结节的反应性星形胶质细胞上;此外,在大鼠的帕金森模型中,VEGF-C可明显增加其血脑屏障的通透性。那么,VEGF-C在MTLE的表达模式是怎样的?其在MTLE发生中的作用及机制又是什么?这些都是值得我们关注和进一步研究的问题。因此,在本研究中,我们首先通过实时定量PCR,Western blot和免疫组化等技术观察VEGF-C及其受体在MTLE患者手术切除的病灶中的表达和细胞定位;然后在匹罗卡品诱导的小鼠MTLE模型中使用Western blot,视频脑电检分别测VEGF-C及其受体在MTLE小鼠癫痫发作的急性期(3小时),潜伏期(1周)和慢性自发性癫痫发作期(3周)的表达和对癫痫发作的影响;同时使用免疫酶联反应(Enzyme-linked immune response,ELISA)检测了VEGF-C及其受体在上述三个时间点对MTLE小鼠海马中血脑屏障开放的标志分子白蛋白(Albumin),白介素1β(IL-1β)IL-6,IL-17和肿瘤坏死因子α(TNF-α)四种和MTLE发生发展密切相关的炎症因子,以及mTOR信号通路(mTOR的激活以p-S6表达增加为标志)的作用。此外,由于在MTLE动物模型中我们发现VEGF-C可上调上述致炎因子,加之我们前期研究发现IL-17在与癫痫发作高度相关的大脑发育障碍中具有重要作用,所以我们还研究了IL-17及其受体IL-17R在MTLE患者手术切除的病灶中的表达和分布。通过上述研究,我们得到如下结果:1.VEGF-C,VEGFR-2和VEGFR-3在MTLE患者致痫灶及脑脊液和血清中的表达水平较对照组明显增高。2.免疫组化及荧光双标结果显示,VEGF-C高表达于MTLE病灶的神经元,反应性星形胶质细胞,以及血管内皮细胞上,VEGFR-2高表达于反应性星形胶质细胞和血管内皮细胞上,而VEGFR3则仅高表达于反应性星形胶质细胞上。3.在匹罗卡品诱导的小鼠MTLE模型中,VEGF-C,VEGFR-2和VEGFR-3在癫痫发作的3 h,1w,和3 w的蛋白表达均显著增高。4.视频脑电监测结果显示,VEGF-C可明显增加MTLE小鼠在上述三个时间点的棘波放电频率和癫痫发作次数,且该效应可被VEGFR-2或者VEGFR-3拮抗剂所阻断。5.VEGF-C上调MTLE小鼠海马在上述一个时间点白蛋白、IL-1β、IL-6、IL-17、TNF-α、p-S6的表达,VEGFR-2拮抗剂可阻断上述效应,VEGFR3拮抗剂可阻断除白蛋白之外的效应。6.IL-17和IL17R在MTLE患者致痫灶中的mRNA和蛋白表达水平较对照组明显增高。7.IL-17和IL-17R高表达于MTLE病灶的神经元,反应性星形胶质细胞和小胶质细胞,以及血管内皮细胞上。我们的研究结果提示,VEGF-C信号通路在MTLE的发生中具有重要作用。
[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.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R742.1

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