【作者】 张春青；

【导师】 杨辉；

【作者基本信息】 第三军医大学 ， 外科学， 2010， 博士

【摘要】 大脑皮质发育障碍(MCD)是药物难治性癫痫的重要病因之一。随着高清影像学技术的普及,MCD的临床检出率不断提高,现已证实约有40%的药物难治性癫痫是由MCD所致,而在小于3岁的儿童药物难治性癫痫病人中更高达80%,因此MCD已成为药物难治性癫痫研究领域内重点和热点的问题。MCD是一类病理学表现类似疾病的总称,主要包括结节性硬化(TSC)、局灶性皮质发育不良(FCD)、多小脑回、无脑回畸形及Sturge-Weber综合征等亚型,其中TSC和FCD是最常见的MCD亚型。尽管MCD根据不同的临床表现具有不同的分类,但都具有共同的组织病理学特点,表现为大脑皮层层状结构和柱状结构紊乱、脑回结构破坏,出现大量异形神经元(DNs)、巨形神经元(GNs)、气球样细胞(BCs)以及巨细胞(GCs)等,这些异常的细胞被称作异构神经元(MNs)。临床资料表明伴有异构神经元的MCD患者更易出现癫痫发作,早期出现耐药。因此,异构神经元在MCD的致痫性中发挥“核心作用”。目前,对异构神经元的病理发生机制尚无明确定论,较为公认的观点认为,异构神经元的产生可能是由胚胎发育期间神经元迁移,成熟和／或细胞死亡过程中出现异常所致。此外,围产期和出生后早期的脑损伤可能也参与了异构神经元的病理发生。总之,目前对MCD及异构神经元病理发生机制所知有限,对涉及其中的关键因子、信号途径仍待阐明。VEGF-C是公认的淋巴系统发生调节因子,通过激活两个特异性受体VEGFR-2, VEGFR-3发挥生物学效应,其中VEGFR-3为VEGF-C的主力型受体。在正常情况下,VEGF-C及VEGFR-3局限性表达于淋巴内皮,而它的另一受体VEGFR-2则在血管内皮和淋巴内皮上均有表达,发挥其促脉管系统发生的作用。由于脑内淋巴系统缺如,既往很少有研究关注VEGF-C在中枢神经系统的作用。直到2006年,有学者发现当小鼠敲除VEGF-C后,表达VEGFR-3受体的神经前体细胞增殖明显受阻,而且胚胎视神经中少突胶质细胞前体细胞选择性缺失,证明VEGF-C信号通路参与了中枢神经系统的生长发育、发挥神经营养功能。近两年来陆续有研究揭示了VEGF-C信号系统在神经系统中的新作用,如VEGF-C/VEGFR-3能够介导胶质前体细胞的增殖,迁移。新近报道脑缺血后,定位于海马齿状回颗粒细胞层最内侧带的细胞高度表达VEGFR-3,这些细胞同时表达与细胞迁移相关的神经前体细胞标记物PSA-NCAM,表明VEGF-C系统参与脑缺血后海马神经发生。此外,脑卒中后,患侧SVZ区内VEGFR-3的表达水平显著上调,表型鉴定实验证实绝大多数表达VEGFR-3的细胞主要为具有旺盛增殖活力的神经祖细胞,另有小部分VEGFR-3阳性细胞为具备迁徙能力的神经前体细胞。在本研究预实验中,我们发现手术切除的MCD标本中VEGF-C mRNA表达上调。那么,VEGF-C及其受体在MCD中的具体表达模式如何?是否表达于异构神经元?高表达的VEGF-C是否参与MCD所致癫痫发作?这些问题值得我们关注。为了阐明VEGF-C在MCD中的作用及其可能的机制,首先,我们利用半定量RT-PCR、western blot、免疫组织化学技术观察了VEGF-C及其受体在MCD(TSC与FCD亚型)临床标本中的表达分布情况；其次,观测了VEGF-C受体信号通路下游相关信号分子的表达活化情况；第三,观察了与VEGF-C高度同源的VEGF-D在MCD中的表达情况；最后,为了直观的了解VEGF-C对MCD致痫效应的影响,我们建立MCD大鼠模型,利用膜片钳全细胞记录技术,观察VEGF-C对MCD大鼠异构神经元兴奋性谷氨酸受体(NMDA受体)的调节效应。通过这些研究,我们得到以下结果：一、VEGF-C及其受体在MCD中的表达分布1.通过RT-PCR, western blot分析,从基因和蛋白水平证明正常皮层中不同程度地表达VEGF-C、VEGFR-2及VEGFR-3;免疫组化结果表明VEGF-C及其受体主要分布于皮层锥体神经元；2.在TSC皮层结节组织匀浆中,VEGF-C及其受体VEGFR-2、VEGFR-3的mRNA及蛋白表达水平较正常对照组明显升高。免疫组化结果显示,VEGF-C, VEGFR-2及VEGFR-3特征性的高表达于DNs、GCs及反应型胶质细胞；免疫荧光双标结果表明：①绝大多数VEGF-C免疫阳性的DNs, GCs共表达神经丝蛋白标记物NF-200；然而未见共表达与另一种神经元标记物NeuN以及星形胶质细胞特异性标志物GFAP共表达；TSC组织中可见CD68阳性的巨噬细胞浸润,但是这些细胞不表达VEGF-C;②VEGFR-2与VEGFR-3的细胞定位表达模式类似,多数VEGFR-2或VEGFR-3免疫阳性的DNs与NF-200共表达,而共表达VEGFR-2 (VEGFR-3)与NF-200的GCs略少,未见VEGFR-2 (VEGFR-3)免疫阳性的DNs, GCs共表达GFAP。3.在另一常见的MCD亚型-FCD中,VEGF-C及受体VEGFR-2、VEGFR-3的mRNA及蛋白表达水平较正常对照组同样显著升高。VEGF-C及其受体高表达于DNs, GNs,BCs及胶质细胞。免疫双标结果显示：①VEGF-C免疫阳性的异构神经元不表达GFAP;与TSC中表达模式不同的是,FCD中的DNs, GNs, BCs能够同时表达VEGF-C与NeuN,提示其具有成熟神经元属性。②VEGFR-3免疫阳性的异构神经元既不表达NF-200,又不表达GFAP,表明其特殊的分子表型。二、MCD病灶内VEGF-C下游相关信号通路的表达活化1. RT-PCR结果表明,MCD中Akt的基因表达上调；利用western blot观察Akt蛋白亚型表达,发现三种Akt亚型的表达均上调,其中Akt-1的表达上调最为明显,表明Akt信号通路参与了MCD的病理生理机制。2.正常皮层及MCD中均可见Bad的表达,且表达水平基本相同；然而,MCD病灶内的磷酸化Bad (p-Bad)表达显著升高,提示MCD中抗凋亡通路活化。3.ERK表达于MCD病灶组织和正常皮层中,呈现特征性的44kDa和42 kDa双条带(ERK 1/2),并可见ERK1/2在MCD中的表达明显上调。三、VEGF-D在MCD中的表达分布1.正常皮层组织匀浆中,可探及VEGF-D mRNA及蛋白表达；免疫组织化学结果表明VEGF-D主要表达于锥体神经元,且免疫反应程度较轻。2.MCD组织匀浆中,VEGF-D mRNA及蛋白表达水平上调。免疫组织化学实验发现：①TSC亚型中VEGF-D表达于多数DNs、GCs及反应性胶质细胞,但免疫阳性强度不一；免疫双标未发现VEGF-D阳性异构神经元与NeuN, NF-200, GFAP及CD68共表达；②FCD亚型中可见VEGF-D表达于DNs, GNs, BCs及反应性胶质细胞,免疫双标结果表明VEGF-D免疫阳性的异构神经元不表达成神经纤维蛋白NF-200,亦不表达成熟星型胶质细胞标记物GFAP。四、VEGF-C对MCD大鼠异构神经元NMDA受体功能的调节效应1.建立MCD动物模型,制备活体脑片后进行膜片钳全细胞记录,结果表明①MCD大鼠异构神经元可产生动作电位,但动作电位形态各异,峰率适应性降低或消失,提示其电生理功能发育不完善；②异构神经元中可记录到evoked-EPSC,但电流形态明显发生改变,通道电流成分混杂,电流面积明显增加,提示MCD异构神经元突触传递效能改变；③MCD异构神经元中NMDA/AMPA受体介导电流的比率显著上调,这种比率改变主要是由NMDA受体电流成分增多引起,表明异构神经元中NMDA受体功能上调。2.MCD大鼠异构神经元中VEGF-C及其受体VEGFR-2的表达明显上调,VEGFR-3的表达亦有增加。3.100μmol/L NMDA作用于异构神经元后,可诱发典型的快反应电流,经过100ng/mL VEGF-C孵育后,相同浓度NMDA诱发的电流显著增大,提示VEGF-C对NMDA受体产生协同作用；当给予VEGFR-2受体阻断剂Ki8751预处理后,这种协同效应被阻断,表明VEGF-C对NMDA受体的调节效应主要是由VEGFR-2受体介导的。综上所述,本研究结果表明：第一、人MCD组织中VEGF-C及其受体VEGFR-2,VEGFR-3表达显著上调,且特征性地高表达于异构神经元；第二、MCD中VEGF-C下游通路中抗凋亡相关信号分子激活,有益地提示VEGF-C可能通过抗凋亡相关信号通路调节MCD内异构神经元的生物学行为；第三、VEGF-D在MCD中的表达上调；第四、VEGF-C通过VEGFR-2增强MCD大鼠异构神经元NMDA受体活性,易化异构神经元的兴奋性。总之,我们的结果提示VEGF-C信号通路参与了MCD的病理生理机制。更多还原

【Abstract】 Malformations of cortical development (MCD) represents a well-recognized cause of intractable epilepsy, with a poor response to the currently available anti-epileptic drugs (AEDs). With the development of high-resolution imageology, the detection rate of MCD is continuously rising during the past decade in clinic.It has been demonstrated that approximately 40 percent of adult patients and more than 80 percent of pediatric with intractable epilepsy are associated with MCD.Thus, the pathogenesis of MCD has became a central concern in the field of intractable epilepsy. MCD encompasses a large spectrum of disorders related to abnormal cortical development, including tuberous sclerosis complex (TSC), focal cortical dysplasia (FCD), microgyria, congenital agyria and Sturge-Weber syndrome. Among these subsets, two of the most common components are TSC and FCD. Despite the fact that these subsets with varied genetic etiologies, anatomic abnormalities, and clinical manifestations, they often share similar histological features such as disorganization or lack of the normal six-layered cortical lamination structure, and the occurrence of malformed neurons (MNs) known as dysplastic neurons (DNs), giant neurons (GNs),balloon cells (BCs) and giant cells (GCs). Mounting clinical evidence has revealed that epilepsy readily occurs in patients with MNs, and appears drug resistance in the early stage, suggesting the dominant role of MNs in epileptogenesis of MCD. Until now, the pathogenesis of MNs remains unclear. A more popular view is that MNs is a malformation due to abnormal migration,mature and cell death during embryonic brain development. Moreover, recent pathological studies on surgical or autoptic human tissue suggest a role for perinatal and early postnatal brain injury in the formation of MNs. In spite of the fact that there are many reports on the subject of MCD, the exact mechanism of this disease has not yet been elucidated.VEGF-C,a well-recognized regulator of lymphangiogenesis, binds to and activates the specific receptors VEGFR-2 and VEGFR-3, and the affinity of VEGFR-3 is much more than that of VEGFR-2. Previous studies show VEGF-C and VEGFR-3 are restrictly expresssed in lymphatic endothelial cells, and VEGFR-2 is expressed in both vascular endothelial cells and lymphatic endothelial cells.Due to the deficiency of the lymphatic system in the brain, few studies have focused on the role of VEGF-C in the CNS. Until to 2006, an important study demonstrats that VEGF-C and VEGFR-3 are present in the neural progenitor cells of Xenopus laevis and mouse embryos. Furthermore, a lack of VEGF-C results in a severe defect in the proliferation of neuronal progenitor cells expressing VEGFR-3, suggesting that VEGF-C is a trophic factor in neuronal progenitor cells in the vertebrate brain. Recently, several studies revealed VEGF-C mediate multiple effects in CNS. For example,activation of the VEGF-C/VEGFR-3 signaling pathway was shown to mediate proliferation and chemotaxis in glial precursor cells. In addition, after a short period of brain ischemia, the innermost layer of granular cells in the hippocampal dental gyrus displayed increased levels of VEGFR-3 expression, these VEGFR-3 immunoreactive cells are also expressed PSA-NCAM, this might be related to neurogenesis in the hippocampus after cerebral ischemia. A recent study showed that the expression of VEGFR-3 increased in subventricular zone, and most of VEGFR-3 positive cells were neural progenitors, were highly proliferative. To our knowledge, there have been no reports about the roles of VEGF-C in in seizure disorders, especially those induced by MCD. In our preliminary experiment,we found that VEGF-C mRNA exhibited high levels in MCD. However, the detailed expression pattern of VEGF-C and it receptors in MCD, and the role of VEGF-C signal system in the epileptogenesis of MCD are unclear.To address these concerns:First,we identified the distribution of VEGF-C system in MCD from patients with medically intractable epilepsy by means of RT-PCR, western blot and immunohistochemistry. Second,we tested the expression and activation of signaling molecule involved in VEGF-C pathway. Third, we investigated the expression and cellular distribution of VEGF-D,which is highly homologous with VEGF-C, in MCD. Fourth, to reveal the role of VEGF-C in the epileptogenesis of MCD, a MCD rat model was employed, and we investigated the effect of VEGF-C on NMDA receptor(NMDAR)of MNs by using whole-cell patch-clamp recordings technique on brain slice of MCD rat model. The results show as following: Ⅰ. Expression and cellular distribution of VEGF-C and VEGF receptors 2 and 3 in MCD1. RT-PCR and western blot analysis showed VEGF-C and its receptors mRNA and protein expression in total homogenates of normal control cortex (CTX); immunohistochemical experiments revealed immunostaining was mainly observed in pyramidal neurons.2. There was a statistically significant increase of VEGF-C, VEGFR-2 and VEGFR-3 mRNA and protein levels in cortical tubers of TSC, in comparison with CTX. The immunostaining results demonstrated that the high-level expression of protein, including VEGF-C and its receptors, was mainly localized within GCs, DNs, and reactive astrocytes. The results of double-labeling immunofluorescence showed:①the co-localization of VEGF-C immunostaining with the neuronal marker NF-200 in majority of DNs and GCs. Intriguingly, we could not detect any co-immunostaining of VEGF-C with another neuronal marker, NeuN, in both DNs and GCs in all cases. Also, GFAP and CD68 immunostaining was not detected in any VEGF-C-positive DNs and GCs.②VEGFR-2 and VEGFR-3 shared a similar cellular distribution pattern in TSC, VEGFR-2 (VEGFR-3) immunoreactivity co-localized with NF-200 in the majority of DNs,the frequency of VEGFR-2 (VEGFR-3) and NF-200 co-expression in GCs was less than that in DNs. VEGFR-2 (VEGFR-3) and GFAP co-immunolabeling was not detected in any DNs and GCs, although prominent co-labeling of reactive astrocytes was noted in all of the TSC cases examined.3. We found an obvious increase of VEGF-C and it receptors mRNA and protein expression in FCD specimens compared with CTX. The high-level expression of protein, including VEGF-C and its receptors, was mainly localized within DNs,GNs,GCs and reactive astrocytes. The results of double-labeling immunofluorescence showed:①GFAP and VEGF-C co-immunolabeling was not detected in any MNs. Interestingly,we detected co-immunostaining of VEGF-C with NeuN, which is usually recognized as a marker of mature neurons, in MNs.②VEGFR-3 and GFAP/NF-200 co-immunolabeling was not observed in any MNs,suggesting a complex phenotype of MNs.Ⅱ. The expression and activation of signaling molecule involved in VEGF-C pathway 1. In comparison with CTX, RT-PCR analysis showed a increase of Akt mRNA levels in MCD specimens. Immunoblots revealed a upward trend in the three types of Akt subsets and the most obvious change was observed in the expression of Akt1.These findings suggested the Akt pathway involved in the pathogenesis of MCD.2. A similar expression levels of Bad was detected in both CTX and MCD specimens, but we detected a significant increase of phosphorylated Bad (P-Bad) in MCD.3. We observed representative immunoblot double-bands of ERK1/2 (42 kDa and 44 kDa) in homogenates from MCD and CTX, and the ERK1/2 protein level in MCD was much high than that in CTX.Ⅲ. Expression and cellular distribution of VEGF-D in MCD1. We detected the VEGF-D mRNA and protein expression in total homogenates of CTX; immunohistochemical results revealed a weak immunostaining was restricted to pyramidal neurons.2. VEGF-D mRNA and protein levels were significantly increased in MCD compared with CTX. The immunostaining results demonstrated:①In TSC specimens, the high-level expression of VEGF-D protein was mainly localized within most of DNs,GCs, and reactive astrocytes; confocal images showed no co-localization of VEGF-D with NeuN, NF-200, GFAP and CD68 in MNs;②In FCD subtype, we found the VEGF-D was highly expressed in DNs,GNs,BCs and reactive astrocytes; the results of double-labeling showed no co-localization of VEGF-D with NF-200 and GFAP in these MNs;V.The modulatory effects of VEGF-C on NMDAR function in MNs from MCD rats model.1. We employed a well-established MCD rat model by in utero irradiation, and In vitro brain slices were obtained from these rats.The results of patch clamp recording showed:①With step-wise depolarization, MNs of MCD responded initially with action potentials (APs),but the shape of APs varied, the spike frequence adaption (SFA) was damaged,even disappeared,suggesting a immature characteristic in electrophysiology.②We successfully observed evoked-EPSC in MNs, and the amplitude and area of evoked-EPSC of MNs were clearly increased,suggesting a complex response comprising multiple inward currents;③The current ratio of NMDAR/AMPAR was increased in MNs compared with pyramidal neurons from normal control, mainly induced by the increase of NMDAR-mediated current, revealing a upregulation of the function of NMDAR.2. The immunostaining results showed there was a significant increase of VEGF-C and VEGFR-2 expression in MNs from MCD rats; the expression level of VEGFR-3 was also statistically increased.3. 100μmol/L NMDA could evoke the inward. After treatment with 100 ng/mL VEGF-C,100μmol/L NMDA caused a pronounced increase of transmembrane current in MNs, the data indicated a synergistic action between VEGF-C and NMDAR in MNs from MCD rats. We also found that Ki8751 (1 nmol/L), a VEGFR-2 inhibitor, abolished the upregulative effects of VEGF-C on NMDA receptor channel, this finding illuminated that the potentiation effects of VEGF-C on NMDAR is mediated by VEGFR-2.In summary, in the present study, we found an obvious increase of VEGF-C and it receptors mRNA and protein expression in MCD specimens compared with the control sample. The high-level expression of protein, including VEGF-C and its receptors, was mainly localized within MNs(DNs,GNs,GCs and BCs). Subsequently, we observed the activation of signaling molecule Akt, Bad, and ERK in MCD, which suggested the VEGF-C miediated anti-apoptotic mechanism might involve in the pathogenesis of MCD. Moreover, our data showed that NMDA receptor-mediated current is markedly potentiated by VEGF-C in the MNs from MCD rats, which suggests that a synergistic action between VEGF-C and glutamate systems exists in MCD. Altogether, these findings illuminate the VEGF-C system involves in the pathogenesis of MCD.更多还原