海马区神经元葡萄糖低代谢在癫痫发生中的作用及机制研究
发布时间：2018-09-08 09:17
【摘要】：癫痫是由于脑内神经元过度异常同步放电所引起的一种慢性脑部疾病,患者表现出具有突然发生、反复发作、一般持续时间较短等特点的中枢神经系统功能障碍。癫痫对于个人、家庭乃至社会均有非常严重的负面作用。在越来越多的临床证据表明发作间期FDG-PET检查在术前致痫灶的定位上具有较好敏感性和准确性。引起癫痫发作的癫痫灶往往表现为葡萄糖代谢减退。很多研究表明反复的癫痫发作能够引起大脑显著缺氧、局部脑区缺血、线粒体功能障碍,这些改变都能够引起组织的葡萄糖利用降低。然而葡萄糖低代谢也能够引起内源性GABAA受体磷酸化的减少而引起神经元抑制网络的不稳定。那么葡萄糖低代谢可能不仅仅是反复癫痫发作的结果而且也可能在癫痫发生过程中起作用。神经元兴奋性和抑制性的不平衡被认为是癫痫发生的原因之一。酸敏感离子通道(ASICs)是一种是电压不敏感的质子门控阳离子通道,已被证明与神经元异常兴奋性密切相关。ASIC1a,ASIC2a和ASIC2b是中枢神经系统中最常表达的ASIC亚基。大量的研究证实大鼠和小鼠模型中应用ASICs的阻断剂阿米洛利可以抑制癫痫发作。遗传学研究显示ASIC1a单核苷酸的基因多态性也与颞叶癫痫发病相关。最新有研究表明ASIC3在颞叶癫痫患者的脑组织内表达升高,而且表达在中间神经元中的ASIC3可能具有抗癫痫作用。我们之前研究表明颞叶癫痫患者和癫痫模型大鼠梨状皮质区ASIC2a表达均升高,并且ASIC2a表达升高可能增加癫痫易感性。能量代谢障碍能够激活多种信号通路和转录因子从而调控基因表达。研究表明在葡萄糖代谢增强的肝细胞癌组织和细胞系中转录因子TFCP2的表达均升高,这提示TFCP2表达可能受到葡萄糖代谢状态的影响。并且TFCP2能够调控ASIC2a的基因表达。那么葡萄糖低代谢是否通过TFCP2调控ASIC2a的表达而促进癫痫发生呢?为了验证这个假设,本研究分为四部分。第一部分:颞叶癫痫患者及癫痫大鼠的低代谢海马脑区中TFCP2及ASIC2a的蛋白表达研究共选取13例MRI结果阴性但PET结果显示单侧海马区葡萄糖低代谢的颞叶癫痫患者的手术切除海马组织标本,以及10例脑外伤患者脑组织标本作为对照。成功制备匹鲁卡品癫痫大鼠模型后,于癫痫后不同时间点进行小动物PET检测和收集癫痫大鼠海马组织。利用蛋白免疫印迹技术检测颞叶癫痫患者及癫痫大鼠的低代谢海马脑区中TFCP2及ASIC2a的蛋白表达情况。结果显示,在癫痫后急性期(2天)和潜伏期(14天)癫痫大鼠海马脑区表现为葡萄糖代谢降低。在癫痫患者和癫痫大鼠的葡萄糖低代谢海马脑区中,TFCP2蛋白表达降低同时ASIC2a表达升高。这提示葡萄糖低代谢出现在癫痫大鼠自发性癫痫发作之前,可能发挥了促进癫痫发生的作用。第二部分:糖缺乏的PC12细胞中TFCP2及ASIC2a的蛋白表达研究对PC12细胞进行低糖培养基、无糖培养基、不同浓度1型葡萄糖转运体抑制剂SFT-31、不同培养环境下(高糖、无糖)不可代谢葡萄糖类似物2-DG处理后,利用蛋白免疫印迹技术检测PC12细胞中TFCP2及ASIC2a的蛋白表达情况。结果显示,低糖、无糖、STF-31、高糖培养条件下2-DG处理的PC12细胞中,TFCP2蛋白表达水平降低和ASIC2a蛋白表达水平升高。在无糖培养条件下,2-DG不引起TFCP2和ASIC2a的蛋白表达变化。这提示葡萄糖缺乏(细胞内可利用糖或糖代谢中间产物的减少)能够直接引起TFCP2和ASIC2a表达变化。第三部分:TFCP2和ASIC2a亚细胞表达定位及TFCP2对ASIC2a表达的调控作用研究通过免疫荧光标记,对癫痫大鼠海马区和糖缺乏的PC12细胞中的TFCP2和ASIC2a蛋白进行定位。用TFCP2-siRNA和过表达TFCP2质粒转染PC12细胞,利用蛋白免疫印迹技术检测PC12细胞中TFCP2及ASIC2a的蛋白表达情况。结果显示,在大鼠CA1锥体神经元和PC12细胞中ASIC2a主要表达于细胞膜和细胞质中,而TFCP2主要表达于细胞质和细胞核中。有趣的是,癫痫大鼠CA1锥体神经元和糖缺乏PC12细胞中,位于细胞核内TFCP2的荧光强度明显减弱、甚至消失。PC12细胞中敲减TFCP2表达引起ASIC2a表达升高,而TFCP2过表达引起ASIC2a表达降低。这提示在整体细胞环境下TFCP2具有负向调控ASIC2表达的功能。第四部分:调控ASIC2a表达后影响神经元内源性兴奋性和癫痫易感性的功能性研究利用阴性对照慢病毒、ASIC2a过表达慢病毒、ASIC2a敲减慢病毒感染器官特异型海马脑片,通过膜片钳系统检测被病毒感染后的CA1椎体神经元的内源性兴奋性。在大鼠CA1区通过脑离体定向注射阴性对照AAV、ASIC2a过表达AAV,待其稳定表达后进行匹鲁卡品诱导的癫痫行为学检测。结果显示,在电生理试验中,ASIC2a过表达神经元在相同电流强度刺激下诱发的动作电位发放个数较阴性对照组增加;同时ASIC2a敲减神经元在相同电流强度刺激下诱发的动作电位发放个数较阴性对照组减少。ASIC2a表达模式改变后CA1椎体神经元的细胞膜输入阻抗无明显变化。这提示ASIC2a过表达引起CA1锥体神经元的内源性兴奋性升高,同时ASIC2a表达减少引起CA1锥体神经元的内源性兴奋性降低。在行为学试验中,海马区ASIC2a过表达组大鼠从注射匹鲁卡品到Racine IV级的时间较对照组明显减少,且达到Racine IV级的比例较对照组明显增多。这提示CA1区ASIC2a过表达引起大鼠癫痫易感性的增加。我们的所有研究结果表明海马区低代谢可以通过抑制TFCP2表达而诱导ASIC2a表达升高,继而增强CA1锥体神经元的内源性兴奋性,并最终导致增加颞叶癫痫的易感性。脑葡萄糖代谢不足可能是诱导癫痫发生的另一种可能的机制。这也为“癫痫发作诱导癫痫发作”假说提供了可能的补充机制。
[Abstract]:Epilepsy is a chronic brain disease caused by excessive abnormal synchronous discharges of neurons in the brain. Patients with epilepsy show abrupt, recurrent and usually short duration of central nervous system dysfunction. Bed evidence suggests that FDG-PET is sensitive and accurate in locating epileptogenic foci before surgery. Epileptic foci that cause seizures are often manifested as impaired glucose metabolism. Many studies have shown that recurrent seizures can cause significant cerebral hypoxia, regional cerebral ischemia, and mitochondrial dysfunction, all of which are altered. Glucose metabolism may not only result from recurrent seizures but also play a role in the development of epilepsy. Acid-sensitive ion channels (ASICs) are voltage-insensitive proton-gated cation channels that have been shown to be closely related to abnormal neuronal excitability. ASIC1a, ASIC2a and ASICC2b are the most frequently expressed ASIC subunits in the central nervous system. Genetic studies have shown that ASIC1a single nucleotide polymorphisms are also associated with temporal lobe epilepsy. Recent studies have shown that ASIC3 expression is elevated in the brain of patients with temporal lobe epilepsy, and ASIC3 expression in intermediate neurons may be present. Previous studies have shown that ASIC2a expression in the piriform cortex of both temporal lobe epilepsy patients and epileptic model rats is elevated, and the elevated expression of ASIC2a may increase the susceptibility to epilepsy. Dysfunction of energy metabolism can activate a variety of signaling pathways and transcription factors to regulate gene expression. The elevated expression of transcription factor TFCP2 in cancer tissues and cell lines suggests that TFCP2 expression may be affected by glucose metabolism. And TFCP2 can regulate the expression of ASIC2a. Does glucose hypometabolism promote epilepsy by regulating the expression of ASIC2a by TFCP2? Part I: Protein expression of TFCP2 and ASIC2a in the hypometabolic hippocampus of temporal lobe epilepsy patients and epileptic rats. Thirteen cases of temporal lobe epilepsy patients with unilateral hypoglycemic hippocampus and 10 cases of brain trauma were selected for surgical excision of hippocampal tissue. After successful preparation of pilocarpine-induced epilepsy rat model, the hippocampal tissues of epileptic rats were collected at different time points after epilepsy. Protein expression of TFCP2 and ASIC2a in the hypometabolic hippocampus of temporal lobe epilepsy patients and epileptic rats were detected by Western blot. In the hippocampus of post-acute (2 days) and latent (14 days) epileptic rats, the glucose metabolism was decreased. In the hippocampus of epileptic patients and epileptic rats, the expression of TFCP2 protein was decreased and the expression of ASIC2a was increased. This suggests that glucose hypometabolism occurs before spontaneous seizures in epileptic rats, and may play a role. Part II: Protein expression of TFCP2 and ASIC2a in PC12 cells with glucose deficiency. PC12 cells were treated with low glucose medium, sugar-free medium, SFT-31, glucose transporter inhibitor of different concentrations, and 2-DG, a glucose analogue that was not metabolized in different culture conditions. The expression of TFCP2 and ASIC2a in PC12 cells was detected by Western blotting. The results showed that the expression of TFCP2 and ASIC2a in PC12 cells treated with 2-DG decreased and ASIC2a increased under low-glucose, sugar-free, STF-31 and high-glucose conditions. Glucose deficiency (reduction of intracellular available glucose or glucose metabolic intermediates) can directly induce changes in the expression of TFCP2 and ASICC2a. Part III: Localization of TFCP2 and ASIC2a subcellular expression and regulation of TFCP2 on ASIC2a expression in hippocampus and PC12 cells with glucose deficiency in epileptic rats by immunofluorescence labeling The protein expression of TFCP2 and ASIC2a in PC12 cells was detected by Western blot. The results showed that ASIC2a was mainly expressed in the cell membrane and cytoplasm of rat CA1 pyramidal neurons and PC12 cells, while TFCP2 was mainly expressed in cytoplasm and cytoplasm. Interestingly, the fluorescence intensity of TFCP2 in the nucleus of CA1 pyramidal neurons and PC12 cells with glucose deficiency in epileptic rats was significantly decreased or even disappeared. The down-regulation of TFCP2 expression in PC12 cells caused the increase of ASIC2a expression, while the over-expression of TFCP2 caused the decrease of ASIC2a expression. This suggests that TFCP2 has a negative regulation in the overall cellular environment. Function of ASIC2 expression. Part IV: Functional study of regulating endogenous excitability and epilepsy susceptibility of neurons after ASIC2a expression. Using negative control lentiviruses, ASIC2a overexpresses lentiviruses, ASIC2a knocks down specific hippocampal slices of virus-infected organs and detects CA1 vertebral neurons by patch clamp system after infection. Endogenous excitability. In CA1 region of rats, AAV was overexpressed by ASIC2a after directional injection of negative control AAV in vitro, and epilepsy induced by pilocarpine was detected after stable expression of AAV. The results showed that in electrophysiological tests, the number of action potential firing induced by over-expressed neurons of ASIC2a under the same current intensity stimulation was negative. At the same time, the number of action potential discharges induced by ASIC2a knockdown neurons stimulated by the same current intensity was lower than that of the negative control group. There was no significant change in membrane input impedance of CA1 vertebral neurons after the change of ASIC2a expression pattern. In behavioral tests, the time from pilocarpine injection to Racine IV was significantly shorter in the overexpression group than in the control group, and the percentage of rats reaching Racine IV was significantly higher than that in the control group. All our findings suggest that hypometabolism in the hippocampus can induce an increase in ASIC2a expression by inhibiting TFCP2 expression, thereby enhancing the intrinsic excitability of CA1 pyramidal neurons, and ultimately increasing the susceptibility to temporal lobe epilepsy. Insufficient glucose metabolism in the brain may be another possibility of inducing epilepsy. This mechanism also provides a possible supplementary mechanism for the hypothesis of epileptic seizure induced seizures.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R742.1
，
本文编号：2230101
[Abstract]:Epilepsy is a chronic brain disease caused by excessive abnormal synchronous discharges of neurons in the brain. Patients with epilepsy show abrupt, recurrent and usually short duration of central nervous system dysfunction. Bed evidence suggests that FDG-PET is sensitive and accurate in locating epileptogenic foci before surgery. Epileptic foci that cause seizures are often manifested as impaired glucose metabolism. Many studies have shown that recurrent seizures can cause significant cerebral hypoxia, regional cerebral ischemia, and mitochondrial dysfunction, all of which are altered. Glucose metabolism may not only result from recurrent seizures but also play a role in the development of epilepsy. Acid-sensitive ion channels (ASICs) are voltage-insensitive proton-gated cation channels that have been shown to be closely related to abnormal neuronal excitability. ASIC1a, ASIC2a and ASICC2b are the most frequently expressed ASIC subunits in the central nervous system. Genetic studies have shown that ASIC1a single nucleotide polymorphisms are also associated with temporal lobe epilepsy. Recent studies have shown that ASIC3 expression is elevated in the brain of patients with temporal lobe epilepsy, and ASIC3 expression in intermediate neurons may be present. Previous studies have shown that ASIC2a expression in the piriform cortex of both temporal lobe epilepsy patients and epileptic model rats is elevated, and the elevated expression of ASIC2a may increase the susceptibility to epilepsy. Dysfunction of energy metabolism can activate a variety of signaling pathways and transcription factors to regulate gene expression. The elevated expression of transcription factor TFCP2 in cancer tissues and cell lines suggests that TFCP2 expression may be affected by glucose metabolism. And TFCP2 can regulate the expression of ASIC2a. Does glucose hypometabolism promote epilepsy by regulating the expression of ASIC2a by TFCP2? Part I: Protein expression of TFCP2 and ASIC2a in the hypometabolic hippocampus of temporal lobe epilepsy patients and epileptic rats. Thirteen cases of temporal lobe epilepsy patients with unilateral hypoglycemic hippocampus and 10 cases of brain trauma were selected for surgical excision of hippocampal tissue. After successful preparation of pilocarpine-induced epilepsy rat model, the hippocampal tissues of epileptic rats were collected at different time points after epilepsy. Protein expression of TFCP2 and ASIC2a in the hypometabolic hippocampus of temporal lobe epilepsy patients and epileptic rats were detected by Western blot. In the hippocampus of post-acute (2 days) and latent (14 days) epileptic rats, the glucose metabolism was decreased. In the hippocampus of epileptic patients and epileptic rats, the expression of TFCP2 protein was decreased and the expression of ASIC2a was increased. This suggests that glucose hypometabolism occurs before spontaneous seizures in epileptic rats, and may play a role. Part II: Protein expression of TFCP2 and ASIC2a in PC12 cells with glucose deficiency. PC12 cells were treated with low glucose medium, sugar-free medium, SFT-31, glucose transporter inhibitor of different concentrations, and 2-DG, a glucose analogue that was not metabolized in different culture conditions. The expression of TFCP2 and ASIC2a in PC12 cells was detected by Western blotting. The results showed that the expression of TFCP2 and ASIC2a in PC12 cells treated with 2-DG decreased and ASIC2a increased under low-glucose, sugar-free, STF-31 and high-glucose conditions. Glucose deficiency (reduction of intracellular available glucose or glucose metabolic intermediates) can directly induce changes in the expression of TFCP2 and ASICC2a. Part III: Localization of TFCP2 and ASIC2a subcellular expression and regulation of TFCP2 on ASIC2a expression in hippocampus and PC12 cells with glucose deficiency in epileptic rats by immunofluorescence labeling The protein expression of TFCP2 and ASIC2a in PC12 cells was detected by Western blot. The results showed that ASIC2a was mainly expressed in the cell membrane and cytoplasm of rat CA1 pyramidal neurons and PC12 cells, while TFCP2 was mainly expressed in cytoplasm and cytoplasm. Interestingly, the fluorescence intensity of TFCP2 in the nucleus of CA1 pyramidal neurons and PC12 cells with glucose deficiency in epileptic rats was significantly decreased or even disappeared. The down-regulation of TFCP2 expression in PC12 cells caused the increase of ASIC2a expression, while the over-expression of TFCP2 caused the decrease of ASIC2a expression. This suggests that TFCP2 has a negative regulation in the overall cellular environment. Function of ASIC2 expression. Part IV: Functional study of regulating endogenous excitability and epilepsy susceptibility of neurons after ASIC2a expression. Using negative control lentiviruses, ASIC2a overexpresses lentiviruses, ASIC2a knocks down specific hippocampal slices of virus-infected organs and detects CA1 vertebral neurons by patch clamp system after infection. Endogenous excitability. In CA1 region of rats, AAV was overexpressed by ASIC2a after directional injection of negative control AAV in vitro, and epilepsy induced by pilocarpine was detected after stable expression of AAV. The results showed that in electrophysiological tests, the number of action potential firing induced by over-expressed neurons of ASIC2a under the same current intensity stimulation was negative. At the same time, the number of action potential discharges induced by ASIC2a knockdown neurons stimulated by the same current intensity was lower than that of the negative control group. There was no significant change in membrane input impedance of CA1 vertebral neurons after the change of ASIC2a expression pattern. In behavioral tests, the time from pilocarpine injection to Racine IV was significantly shorter in the overexpression group than in the control group, and the percentage of rats reaching Racine IV was significantly higher than that in the control group. All our findings suggest that hypometabolism in the hippocampus can induce an increase in ASIC2a expression by inhibiting TFCP2 expression, thereby enhancing the intrinsic excitability of CA1 pyramidal neurons, and ultimately increasing the susceptibility to temporal lobe epilepsy. Insufficient glucose metabolism in the brain may be another possibility of inducing epilepsy. This mechanism also provides a possible supplementary mechanism for the hypothesis of epileptic seizure induced seizures.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R742.1
，
本文编号：2230101
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