P2X7受体在大脑皮质发育障碍癫痫中的作用机制研究
发布时间：2018-07-22 17:02
【摘要】：由神经细胞增殖、迁移、分化异常所导致的皮质发育障碍(Malformation of cortical development,MCD)是癫痫形成的重要病理生理基础,而且是导致其药物难以控制的重要原因之一。近年来随着高清影像学技术的普及,MCD的临床检出率不断提高,为进一步研究癫痫提供了重要的临床条件,也使得这一疾病日益成为癫痫领域最活跃的研究热点之一,故而对MCD的发生及致痫机制的研究将会推进对药物难治性癫痫发病机制的新认识及防治策略的革新,并且对改善癫痫患者生存质量有重要现实意义。MCD的最常见的临床分型包括局灶性皮质发育不良(FCD)及结节性硬化(TSC)。其典型病理学特征是皮层板层结构紊乱、病灶内出现异常发育的异构神经元如气球样细胞(Balloon cells,BC)、异形神经元(Dysmorphic neuron,DN),巨大神经元(Giant neuron,GN)等。我们及其他学者的临床研究显示具有异形神经元的FCD II型(皮层结构紊乱+异构神经元)较I型(仅有皮层结构紊乱)患者癫痫发病年龄更早、发作频率更高、发作程度更严重、药物难治性比例更高。但MCD的致痫机制十分复杂,至今尚不完全清楚,随着近年研究的深入,无菌性炎症与癫痫的关系越来越受到关注,炎症相关的异常电活动在癫痫的发生、发展及预后中所起的关键作用。深入研究炎症信号在MCD致痫灶中的作用,将有助于人们进一步认识和阐明MCD的致痫机制。在本研究中,我们以MCD临床手术切除的标本为研究对象,并建立了MCD动物模型,初步研究了FCD和TSC皮层病灶以及动物模型中P2X7R-Caspase1-IL1β信号系统的表达分布,分析了P2X7R及其介导的炎症信号系统在致痫灶中的潜在作用。主要结果如下:一、嘌呤能离子通道受体7(Purinergic ligand-gated ion channel 7 receptor,P2X7R)在MCD患者大脑皮层中的表达分布1.应用Real-time PCR及Western blot技术,我们评价了P2X7R m RNA及蛋白在CTX及FCD临床标本中的表达情况。结果显示,P2X7R的m RNA水平高于正常对照CTX(P0.05);在FCD各个亚型中,FCD IIa和FCD IIb型的m RNA水平明显高于FCD Ia,差异有统计学意义(P0.05),而FCD IIa和FCD IIb的P2X7R m RNA表达水平差异未见统计学意义;Western-blot检测P2X7R结果同样显示,在临床FCD标本中,P2X7R的蛋白水平明显高于CTX正常对照(P0.05),并且FCDII型中的蛋白水平明显高于FCD Ia(P0.05)。2.利用免疫组化及荧光双标技术,我们进一步分析了P2X7R在CTX和FCD组织中的定位情况,免疫组化显示,P2X7R在CTX标本中蛋白表达呈弱阳性,在灰质区域主要表达在神经细胞中,而在白质区域,可于少量胶质细胞样细胞上呈较弱表达;在FCD Ia组织中,P2X7R呈现出中到强表达于灰质及灰、白质交界区的微柱状排列的肥大神经细胞中,也表达于灰、白质交界区的部分胶质细胞样细胞中;而在FCD IIa和FCD IIb标本中,P2X7R强表达于75%±5.6%的DN和64%±6.7%的BC中。并且值得注意的是,在FCD标本中,细胞大小、形态大致正常的神经元中,P2X7R的表达也明显高于CTX组。免疫荧光双标结果显示,在FCD标本中,P2X7R表现出较强的免疫活性,在异形神经元(dysmorphic neuron,DN)和气球样细胞(Balloon cell,BC)中均有表达,与神经元标志物Neu N共标,同时与大部分(80%)活化的星形胶质细胞(GFAP)和小胶质细胞(HLA)共标。3.我们同时评价了P2X7R m RNA及蛋白在TSC临床标本致痫结节中的表达情况,结果显示,P2X7R的m RNA水平较正常对照CTX显著增高,其差异有统计学意义(P0.05);Western-blot检测P2X7R结果同样显示,在临床TSC标本中,P2X7R的蛋白水平明显高于CTX正常对照(P0.05)。免疫组化结果中,相对于CTX标本,P2X7R在TSC标本中强表达于超过90%的DN中;免疫荧光双标结果显示,在TSC标本中,P2X7R强表达于异形神经元细胞中(DN,BC,GN),其免疫阳性细胞可与神经元标志物NF200和星形胶质细胞GFAP共标。二、IL-1β及其受体在FCD中的表达分布对临床FCD标本的研究显示,IL-1β/IL-1R1的m RNA水平高于正常对照CTX(P0.05);在FCD各个亚型中,FCD IIa和FCD IIb型的IL-1β/IL-1R1 m RNA水平明显高于FCD Ia(P0.05),而在FCD IIa和FCD IIb的比较中,二者m RNA水平差异未见统计学意义;Western-blot检测IL-1β/IL-1R1结果同样显示,在临床FCD标本中,IL-1β/IL-1R1二者的蛋白水平明显高于CTX正常对照(P0.05),并且FCDII型中的蛋白水平明显高于FCD Ia(P0.05)。免疫组化显示,IL-1β及IL-1R1在CTX标本中免疫组化染色呈弱阳性,主要在神经元样细胞和少量星形胶质细胞样细胞上发现较弱表达;在FCD Ia组织中,IL-1β/IL-1R1呈现出中到强表达于灰质及灰、白质交界区的微柱状排列的肥大神经细胞中,也表达于灰、白质交界区的部分星形胶质细胞样细胞中;而在FCD IIa和FCD IIb标本中,IL-1β强表达于89%±6.2%的DN(n=650)和84%±5.7%的BC(n=255)中。免疫荧光双标结果显示,在FCD标本中,IL-1β/IL-1R1表现出较强的免疫活性,表达在异形神经元DN和BC,与神经元标志物Neu N共标,同时与大部分(80%)活化的星形胶质细胞(GFAP)和小胶质细胞(HLA)共标,此表达分布与P2X7R类似。三、P2X7受体在烷基化物甲基偶氮甲醇(methylazoxymethanol acetate,MAM)模型中的表达及蛋白分布1.MCD模型大鼠皮质发育障碍模型基本特点我们应用烷基化物甲基偶氮甲醇(methylazoxymethanol acetate,MAM)给予孕鼠腹腔注射,诱导仔鼠出现类似临床病理改变的皮质发育障碍,从而对MCD进行动物模拟。结果提示MAM大鼠皮质发育障碍动物模型,仔鼠出现小头畸形,自主活动减少,对外界刺激反应较迟钝,爬行及躲避性动作灵活性下降,摄食能力下降,体重减轻,主要病理表现为脑皮质变薄,板层结构不清,细胞排列紊乱,神经元形态异常,神经元胞体增大,正常极性消失,于分子层可见众多异位的神经元,呈团块样聚集,并可见病理性结节,具有FCDII型的病理改变;2.P2X7受体m RNA在CTX及MAM大鼠MCD模型皮层中的表达情况应用Real-time PCR、免疫组化方法,对MAM大鼠皮质发育障碍模型和正常对照进行了研究,结果发现,在MAM皮质发育障碍模型不同年龄段(2,4,6周)大鼠标本中,同年龄段组内比较,P2X7 m RNA在MAM皮质发育障碍大鼠皮层中表达明显高于正常对照组(CTX);应用免疫组化方法对比了P2X7R、Caspase-1、IL-1β在MAM模型大鼠脑组织中的表达分布,结果显示P2X7R在大鼠CTX正常对照标本中呈弱到中等强度表达,但仅分布在少量(22.9%±3.4%,n=379)的神经元样细胞中、胶质细胞样细胞上发现较弱表达;而在MAM皮质发育障碍组织中,皮层细胞排列紊乱,P2X7R中到强表达并广泛分布于灰质及灰、白质交界区中排列紊乱、形态异常的“异形神经元”和星形胶质细胞中;同样IL-1β、Caspase-1在MAM皮质发育障碍大鼠皮层中的m RNA及蛋白表达明显高于CTX正常对照标本;MAM皮质发育障碍模型大鼠的IL-1β表达强度及免疫阳性细胞数量明显高于对照组。结论如下:1.我们首次从m RNA和蛋白水平分析了P2X7R在CTX和FCD中的表达情况。结果显示,FCD患者P2X7R的m RNA及蛋白表达显著高于正常对照CTX;FCD IIa和FCD IIb的m RNA及蛋白水平明显高于FCD Ia。并且在FCD Ia组织中,P2X7R强表达于灰质及灰、白质交界区的微柱状排列的肥大神经细胞,也表达于灰、白质交界区的部分胶质细胞样细胞中;而在FCD IIa和FCD IIb标本中,P2X7R强表达于DN和BC中,与神经元标志物Neu N共标,同时与大部分(80%)活化的星形胶质细胞(GFAP)和小胶质细胞(HLA)共标。2.TSC患者致痫灶中P2X7R m RNA及蛋白表达显著高于对照组,并且相对于CTX标本,P2X7R在TSC标本中强表达于超过90%的DN中;与神经元标志物NF200及星形胶质细胞标志物GFAP共标。3.在FCD患者致痫灶内,IL-1β及其受体IL-1R1及Caspase-1 m RNA及蛋白表达均显著高于正常对照组。4.最后我们采用孕鼠腹腔注射MAM诱导MCD动物模型的方法,初步验证了P2X7R对癫痫发作的作用,结果显示P2X7R激动剂Bz ATP可以使癫痫发作时间明显提前,易化了匹鲁卡品诱导的癫痫发作。综合上述结果,我们推断:P2X7R及其介导的Caspase1-I/L1β信号通路,在MCD癫痫发作的过程中发挥重要致痫作用。
[Abstract]:Cortical dysplasia (Malformation of cortical development, MCD), which is caused by the proliferation, migration and differentiation of nerve cells, is an important pathophysiological basis for the formation of epilepsy, and is one of the important reasons for the difficult control of its drugs. In recent years, with the popularization of high definition imaging technology, the clinical detection rate of MCD has been continuously improved. The further study of epilepsy provides important clinical conditions and makes the disease one of the most active research hot spots in the field of epilepsy. Therefore, the study of the pathogenesis of MCD and the mechanism of epilepsy will promote the new understanding of the mechanism of drug refractory epilepsy and the innovation of the prevention and treatment strategies, and improve the quality of life of the epileptic patients. The most common clinical types of important practical.MCD include focal cortical dysplasia (FCD) and nodular sclerosis (TSC). The typical pathological features are the disorder of the cortical lamellar structure, and the abnormal development of the isomerism neurons such as the Balloon cells (BC), the heteromorphic neurons (Dysmorphic neuron, DN), and the giant nerve. Giant neuron, GN, et al. Our and other scholars' clinical studies show that FCD II (cortical disorder + isomeric neurons) with heteromorphic neurons (cortical structural disorders + isomeric neurons) are older than those of the I type (only cortical disorders), with more frequent episodes, more severe episodes and higher drug refractory proportions. But the mechanism of MCD is very complex. The relationship between aseptic inflammation and epilepsy is becoming more and more concerned with recent research. The key role of abnormal electrical activity related to inflammation in the occurrence, development and prognosis of epilepsy. The in-depth study of the role of inflammatory signals in MCD epileptic foci will help people to further understand and clarify MCD In this study, in this study, we studied the specimens removed by MCD and established the MCD animal model. We preliminarily studied the expression and distribution of the P2X7R-Caspase1-IL1 beta signal system in the FCD and TSC cortical lesions and the animal models, and analyzed the potential role of P2X7R and its mediated inflammatory signal system in the epileptic foci. The main results are as follows: (1) the expression distribution of the purine energy ion channel receptor 7 (Purinergic ligand-gated ion channel 7 receptor, P2X7R) in the cerebral cortex of the MCD patients 1. application of Real-time PCR and Western blot technique. The level of M RNA in FCD IIa and FCD IIb was significantly higher than that of FCD Ia in the FCD subtypes of FCD, and there was no significant difference in the level of M RNA in FCD IIa and FCD IIb. The protein level in FCDII was significantly higher than that of CTX (P0.05), and the protein level in FCDII was significantly higher than that of FCD Ia (P0.05).2. using immunohistochemistry and fluorescence double labeling technique. We further analyzed the localization of P2X7R in CTX and FCD tissues. Immunohistochemistry showed that P2X7R protein expression in the CTX specimens was weakly positive, and the main expression in the gray matter region was in the deity. In the cells, in the white matter region, it can be weakly expressed on a small number of glial cell like cells; in FCD Ia tissue, P2X7R appears to be strongly expressed in gray matter and ash, and in the microcolumnar hypertrophic neurons of the white matter junction, also in some glial cell like cells of the gray matter and the white matter junction; and in the FCD IIa and FCD IIb standard. In this case, P2X7R was strongly expressed in 75% + 5.6% DN and 64% + 6.7% BC. And it is worth noting that in the FCD specimens, the expression of P2X7R was significantly higher than that in the CTX group. The results of immunofluorescence double labeling showed that P2X7R showed strong immune activity in the FCD specimens, and in the heteromorphic neurons (dysmorphic neu). Ron, DN) and balloon like cells (Balloon cell, BC) were expressed, CO labeled with the neuronal marker Neu N, and co labeled with most (80%) activated astrocytes (GFAP) and microglia (HLA). We also evaluated the expression of P2X7R m and protein in the epileptic nodules on the bed specimens. Compared with normal control CTX, the difference was statistically significant (P0.05); Western-blot test P2X7R results also showed that in the clinical TSC specimens, the protein level of P2X7R was significantly higher than that of the normal CTX control (P0.05). In the immunohistochemical results, the P2X7R in the TSC specimen was strongly expressed in more than 90% of the DN; double immunofluorescent labeling. The results showed that in the TSC specimens, P2X7R was strongly expressed in the heteromorphic neurons (DN, BC, GN), and the immunoreactive cells were co labeled with the neuronal marker NF200 and astrocyte GFAP. Two, the expression distribution of IL-1 beta and its receptor in FCD showed that the IL-1 beta /IL-1R1 was higher than the normal control. In the various subtypes of FCD, the IL-1 beta /IL-1R1 m RNA levels of FCD IIa and FCD IIb were significantly higher than FCD Ia (P0.05). Normal control (P0.05), and the protein level in type FCDII was significantly higher than that of FCD Ia (P0.05). Immunohistochemical staining showed that IL-1 beta and IL-1R1 were weakly positive in immunohistochemical staining in CTX specimens, mainly in the neuron like cells and a small number of astrocyte like cells. In FCD Ia tissue, IL-1 beta /IL-1R1 presented a medium to strong expression. In the microcolumnar hypertrophic neurons of gray matter and gray matter and white matter junction, it is also expressed in some astrocytes like cells in the gray and white matter junction area; and in FCD IIa and FCD IIb specimens, IL-1 beta is strongly expressed in 89% + 6.2% DN (n=650) and 84% + 5.7% BC (n= 255). The immunofluorescence double standard results show IL-1 beta in FCD specimens. IL-1R1 showed strong immune activity, expressed in heteromorphic neurons DN and BC, CO labeled with neuron marker Neu N, and co labeled with most (80%) activated astrocytes (GFAP) and microglia (HLA). This expression was similar to P2X7R. Three, P2X7 receptor was in alkylate methyl azo methanol (methylazoxymethanol acetate, MAM). The expression and protein distribution in the model 1.MCD model rat cortical dysplasia model basic characteristics we use the alkyl methyl azo methanol (methylazoxymethanol acetate, MAM) to give pregnant rats intraperitoneal injection to induce the offspring to appear similar to the clinicopathological changes of cortical development obstacle, thus the animal simulation of MCD. The results suggest that MAM is large. The animal model of rat cortical development disorder showed small head deformity, decreased autonomic activity, slow response to external stimulation, decreased flexibility of crawling and avoidance action, decreased feeding ability, and weight loss. The main pathological manifestations were the thinning of the cortex, the indistinct structure of the lamina, the disorder of the cells, the abnormal neuronal morphology, and the enlargement of the cell body. The normal polarity disappeared, and a large number of heterotopic neurons were found on the molecular layer. The pathological nodules were clustered, and pathological nodules were seen with FCDII type pathological changes. The expression of the 2.P2X7 receptor m RNA in the MCD model cortex of CTX and MAM rats was used in Real-time PCR, immunohistochemical method, and the model of cortical dysplasia and normal control in MAM rats. The results showed that the expression of P2X7 m RNA in the cortex of MAM cortical dysplasia rats was significantly higher than that in the normal control group (CTX) in the same age group (CTX) in the different age group (2,4,6 weeks) of the MAM cortical dysplasia model, and the immunohistochemical method was used to compare the P2X7R, Caspase-1, IL-1 beta in the brain tissue of the MAM model rats. The results showed that P2X7R was weak to medium intensity expression in CTX normal control specimens of rats, but only in a small number of neuron like cells (22.9% + 3.4%, n=379), the weak expression was found on the glial cell like cells, while in the MAM cortical dysplasia tissue, the peri layer cells were arranged in disorder, and the expression of P2X7R was strongly expressed and widely distributed in the MAM cortical dysplasia. In gray matter and ash and white matter junction, the disorder, morphologically abnormal "heteromorphic neurons" and astrocytes, the same IL-1 beta, Caspase-1 in the cortex of MAM cortical dysplasia rats, m RNA and protein expression are significantly higher than those of CTX normal controls; the expression intensity of IL-1 beta and immunoreactive cells in the rat model of MAM cortical dysplasia model The number of P2X7R in CTX and FCD was analyzed for the first time from m RNA and protein levels. The results showed that the m RNA and protein expression of P2X7R in FCD patients was significantly higher than that of normal control CTX. The microcolumnar hypertrophic neurons in gray matter and white matter junction are also expressed in some glial cell like cells in the gray and white matter junction; in FCD IIa and FCD IIb specimens, P2X7R is strongly expressed in DN and BC, CO marked with the neuronal marker Neu N, and with most (80%) activated astrocytes (GFAP) and microglia. The expression of P2X7R m RNA and protein in the epileptic foci of.2.TSC patients was significantly higher than that of the control group. Compared with the CTX specimens, P2X7R was strongly expressed in more than 90% of DN in TSC specimens, and the.3. was in the epileptic focus of the patients with neuronal markers NF200 and astrocyte markers GFAP. The expression of A and protein were significantly higher than that of the normal control group.4.. Finally, we used the method of intraperitoneal injection of MAM to induce the MCD animal model in the pregnant rats. The effect of P2X7R on epileptic seizures was preliminarily verified. The results showed that the P2X7R agonist Bz ATP could lead to the seizure time obviously ahead of time and the seizures induced by bluka. We conclude that P2X7R and its Caspase1-I/L1 beta signaling pathway play an important role in epileptic seizure in MCD.
【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
本文编号：2138152
[Abstract]:Cortical dysplasia (Malformation of cortical development, MCD), which is caused by the proliferation, migration and differentiation of nerve cells, is an important pathophysiological basis for the formation of epilepsy, and is one of the important reasons for the difficult control of its drugs. In recent years, with the popularization of high definition imaging technology, the clinical detection rate of MCD has been continuously improved. The further study of epilepsy provides important clinical conditions and makes the disease one of the most active research hot spots in the field of epilepsy. Therefore, the study of the pathogenesis of MCD and the mechanism of epilepsy will promote the new understanding of the mechanism of drug refractory epilepsy and the innovation of the prevention and treatment strategies, and improve the quality of life of the epileptic patients. The most common clinical types of important practical.MCD include focal cortical dysplasia (FCD) and nodular sclerosis (TSC). The typical pathological features are the disorder of the cortical lamellar structure, and the abnormal development of the isomerism neurons such as the Balloon cells (BC), the heteromorphic neurons (Dysmorphic neuron, DN), and the giant nerve. Giant neuron, GN, et al. Our and other scholars' clinical studies show that FCD II (cortical disorder + isomeric neurons) with heteromorphic neurons (cortical structural disorders + isomeric neurons) are older than those of the I type (only cortical disorders), with more frequent episodes, more severe episodes and higher drug refractory proportions. But the mechanism of MCD is very complex. The relationship between aseptic inflammation and epilepsy is becoming more and more concerned with recent research. The key role of abnormal electrical activity related to inflammation in the occurrence, development and prognosis of epilepsy. The in-depth study of the role of inflammatory signals in MCD epileptic foci will help people to further understand and clarify MCD In this study, in this study, we studied the specimens removed by MCD and established the MCD animal model. We preliminarily studied the expression and distribution of the P2X7R-Caspase1-IL1 beta signal system in the FCD and TSC cortical lesions and the animal models, and analyzed the potential role of P2X7R and its mediated inflammatory signal system in the epileptic foci. The main results are as follows: (1) the expression distribution of the purine energy ion channel receptor 7 (Purinergic ligand-gated ion channel 7 receptor, P2X7R) in the cerebral cortex of the MCD patients 1. application of Real-time PCR and Western blot technique. The level of M RNA in FCD IIa and FCD IIb was significantly higher than that of FCD Ia in the FCD subtypes of FCD, and there was no significant difference in the level of M RNA in FCD IIa and FCD IIb. The protein level in FCDII was significantly higher than that of CTX (P0.05), and the protein level in FCDII was significantly higher than that of FCD Ia (P0.05).2. using immunohistochemistry and fluorescence double labeling technique. We further analyzed the localization of P2X7R in CTX and FCD tissues. Immunohistochemistry showed that P2X7R protein expression in the CTX specimens was weakly positive, and the main expression in the gray matter region was in the deity. In the cells, in the white matter region, it can be weakly expressed on a small number of glial cell like cells; in FCD Ia tissue, P2X7R appears to be strongly expressed in gray matter and ash, and in the microcolumnar hypertrophic neurons of the white matter junction, also in some glial cell like cells of the gray matter and the white matter junction; and in the FCD IIa and FCD IIb standard. In this case, P2X7R was strongly expressed in 75% + 5.6% DN and 64% + 6.7% BC. And it is worth noting that in the FCD specimens, the expression of P2X7R was significantly higher than that in the CTX group. The results of immunofluorescence double labeling showed that P2X7R showed strong immune activity in the FCD specimens, and in the heteromorphic neurons (dysmorphic neu). Ron, DN) and balloon like cells (Balloon cell, BC) were expressed, CO labeled with the neuronal marker Neu N, and co labeled with most (80%) activated astrocytes (GFAP) and microglia (HLA). We also evaluated the expression of P2X7R m and protein in the epileptic nodules on the bed specimens. Compared with normal control CTX, the difference was statistically significant (P0.05); Western-blot test P2X7R results also showed that in the clinical TSC specimens, the protein level of P2X7R was significantly higher than that of the normal CTX control (P0.05). In the immunohistochemical results, the P2X7R in the TSC specimen was strongly expressed in more than 90% of the DN; double immunofluorescent labeling. The results showed that in the TSC specimens, P2X7R was strongly expressed in the heteromorphic neurons (DN, BC, GN), and the immunoreactive cells were co labeled with the neuronal marker NF200 and astrocyte GFAP. Two, the expression distribution of IL-1 beta and its receptor in FCD showed that the IL-1 beta /IL-1R1 was higher than the normal control. In the various subtypes of FCD, the IL-1 beta /IL-1R1 m RNA levels of FCD IIa and FCD IIb were significantly higher than FCD Ia (P0.05). Normal control (P0.05), and the protein level in type FCDII was significantly higher than that of FCD Ia (P0.05). Immunohistochemical staining showed that IL-1 beta and IL-1R1 were weakly positive in immunohistochemical staining in CTX specimens, mainly in the neuron like cells and a small number of astrocyte like cells. In FCD Ia tissue, IL-1 beta /IL-1R1 presented a medium to strong expression. In the microcolumnar hypertrophic neurons of gray matter and gray matter and white matter junction, it is also expressed in some astrocytes like cells in the gray and white matter junction area; and in FCD IIa and FCD IIb specimens, IL-1 beta is strongly expressed in 89% + 6.2% DN (n=650) and 84% + 5.7% BC (n= 255). The immunofluorescence double standard results show IL-1 beta in FCD specimens. IL-1R1 showed strong immune activity, expressed in heteromorphic neurons DN and BC, CO labeled with neuron marker Neu N, and co labeled with most (80%) activated astrocytes (GFAP) and microglia (HLA). This expression was similar to P2X7R. Three, P2X7 receptor was in alkylate methyl azo methanol (methylazoxymethanol acetate, MAM). The expression and protein distribution in the model 1.MCD model rat cortical dysplasia model basic characteristics we use the alkyl methyl azo methanol (methylazoxymethanol acetate, MAM) to give pregnant rats intraperitoneal injection to induce the offspring to appear similar to the clinicopathological changes of cortical development obstacle, thus the animal simulation of MCD. The results suggest that MAM is large. The animal model of rat cortical development disorder showed small head deformity, decreased autonomic activity, slow response to external stimulation, decreased flexibility of crawling and avoidance action, decreased feeding ability, and weight loss. The main pathological manifestations were the thinning of the cortex, the indistinct structure of the lamina, the disorder of the cells, the abnormal neuronal morphology, and the enlargement of the cell body. The normal polarity disappeared, and a large number of heterotopic neurons were found on the molecular layer. The pathological nodules were clustered, and pathological nodules were seen with FCDII type pathological changes. The expression of the 2.P2X7 receptor m RNA in the MCD model cortex of CTX and MAM rats was used in Real-time PCR, immunohistochemical method, and the model of cortical dysplasia and normal control in MAM rats. The results showed that the expression of P2X7 m RNA in the cortex of MAM cortical dysplasia rats was significantly higher than that in the normal control group (CTX) in the same age group (CTX) in the different age group (2,4,6 weeks) of the MAM cortical dysplasia model, and the immunohistochemical method was used to compare the P2X7R, Caspase-1, IL-1 beta in the brain tissue of the MAM model rats. The results showed that P2X7R was weak to medium intensity expression in CTX normal control specimens of rats, but only in a small number of neuron like cells (22.9% + 3.4%, n=379), the weak expression was found on the glial cell like cells, while in the MAM cortical dysplasia tissue, the peri layer cells were arranged in disorder, and the expression of P2X7R was strongly expressed and widely distributed in the MAM cortical dysplasia. In gray matter and ash and white matter junction, the disorder, morphologically abnormal "heteromorphic neurons" and astrocytes, the same IL-1 beta, Caspase-1 in the cortex of MAM cortical dysplasia rats, m RNA and protein expression are significantly higher than those of CTX normal controls; the expression intensity of IL-1 beta and immunoreactive cells in the rat model of MAM cortical dysplasia model The number of P2X7R in CTX and FCD was analyzed for the first time from m RNA and protein levels. The results showed that the m RNA and protein expression of P2X7R in FCD patients was significantly higher than that of normal control CTX. The microcolumnar hypertrophic neurons in gray matter and white matter junction are also expressed in some glial cell like cells in the gray and white matter junction; in FCD IIa and FCD IIb specimens, P2X7R is strongly expressed in DN and BC, CO marked with the neuronal marker Neu N, and with most (80%) activated astrocytes (GFAP) and microglia. The expression of P2X7R m RNA and protein in the epileptic foci of.2.TSC patients was significantly higher than that of the control group. Compared with the CTX specimens, P2X7R was strongly expressed in more than 90% of DN in TSC specimens, and the.3. was in the epileptic focus of the patients with neuronal markers NF200 and astrocyte markers GFAP. The expression of A and protein were significantly higher than that of the normal control group.4.. Finally, we used the method of intraperitoneal injection of MAM to induce the MCD animal model in the pregnant rats. The effect of P2X7R on epileptic seizures was preliminarily verified. The results showed that the P2X7R agonist Bz ATP could lead to the seizure time obviously ahead of time and the seizures induced by bluka. We conclude that P2X7R and its Caspase1-I/L1 beta signaling pathway play an important role in epileptic seizure in MCD.
【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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