海馬區(qū)神經(jīng)元葡萄糖低代謝在癲癇發(fā)生中的作用及機(jī)制研究
發(fā)布時(shí)間：2018-09-08 09:17
【摘要】：癲癇是由于腦內(nèi)神經(jīng)元過(guò)度異常同步放電所引起的一種慢性腦部疾病,患者表現(xiàn)出具有突然發(fā)生、反復(fù)發(fā)作、一般持續(xù)時(shí)間較短等特點(diǎn)的中樞神經(jīng)系統(tǒng)功能障礙。癲癇對(duì)于個(gè)人、家庭乃至社會(huì)均有非常嚴(yán)重的負(fù)面作用。在越來(lái)越多的臨床證據(jù)表明發(fā)作間期FDG-PET檢查在術(shù)前致癇灶的定位上具有較好敏感性和準(zhǔn)確性。引起癲癇發(fā)作的癲癇灶往往表現(xiàn)為葡萄糖代謝減退。很多研究表明反復(fù)的癲癇發(fā)作能夠引起大腦顯著缺氧、局部腦區(qū)缺血、線粒體功能障礙,這些改變都能夠引起組織的葡萄糖利用降低。然而葡萄糖低代謝也能夠引起內(nèi)源性GABAA受體磷酸化的減少而引起神經(jīng)元抑制網(wǎng)絡(luò)的不穩(wěn)定。那么葡萄糖低代謝可能不僅僅是反復(fù)癲癇發(fā)作的結(jié)果而且也可能在癲癇發(fā)生過(guò)程中起作用。神經(jīng)元興奮性和抑制性的不平衡被認(rèn)為是癲癇發(fā)生的原因之一。酸敏感離子通道(ASICs)是一種是電壓不敏感的質(zhì)子門控陽(yáng)離子通道,已被證明與神經(jīng)元異常興奮性密切相關(guān)。ASIC1a,ASIC2a和ASIC2b是中樞神經(jīng)系統(tǒng)中最常表達(dá)的ASIC亞基。大量的研究證實(shí)大鼠和小鼠模型中應(yīng)用ASICs的阻斷劑阿米洛利可以抑制癲癇發(fā)作。遺傳學(xué)研究顯示ASIC1a單核苷酸的基因多態(tài)性也與顳葉癲癇發(fā)病相關(guān)。最新有研究表明ASIC3在顳葉癲癇患者的腦組織內(nèi)表達(dá)升高,而且表達(dá)在中間神經(jīng)元中的ASIC3可能具有抗癲癇作用。我們之前研究表明顳葉癲癇患者和癲癇模型大鼠梨狀皮質(zhì)區(qū)ASIC2a表達(dá)均升高,并且ASIC2a表達(dá)升高可能增加癲癇易感性。能量代謝障礙能夠激活多種信號(hào)通路和轉(zhuǎn)錄因子從而調(diào)控基因表達(dá)。研究表明在葡萄糖代謝增強(qiáng)的肝細(xì)胞癌組織和細(xì)胞系中轉(zhuǎn)錄因子TFCP2的表達(dá)均升高,這提示TFCP2表達(dá)可能受到葡萄糖代謝狀態(tài)的影響。并且TFCP2能夠調(diào)控ASIC2a的基因表達(dá)。那么葡萄糖低代謝是否通過(guò)TFCP2調(diào)控ASIC2a的表達(dá)而促進(jìn)癲癇發(fā)生呢?為了驗(yàn)證這個(gè)假設(shè),本研究分為四部分。第一部分:顳葉癲癇患者及癲癇大鼠的低代謝海馬腦區(qū)中TFCP2及ASIC2a的蛋白表達(dá)研究共選取13例MRI結(jié)果陰性但PET結(jié)果顯示單側(cè)海馬區(qū)葡萄糖低代謝的顳葉癲癇患者的手術(shù)切除海馬組織標(biāo)本,以及10例腦外傷患者腦組織標(biāo)本作為對(duì)照。成功制備匹魯卡品癲癇大鼠模型后,于癲癇后不同時(shí)間點(diǎn)進(jìn)行小動(dòng)物PET檢測(cè)和收集癲癇大鼠海馬組織。利用蛋白免疫印跡技術(shù)檢測(cè)顳葉癲癇患者及癲癇大鼠的低代謝海馬腦區(qū)中TFCP2及ASIC2a的蛋白表達(dá)情況。結(jié)果顯示,在癲癇后急性期(2天)和潛伏期(14天)癲癇大鼠海馬腦區(qū)表現(xiàn)為葡萄糖代謝降低。在癲癇患者和癲癇大鼠的葡萄糖低代謝海馬腦區(qū)中,TFCP2蛋白表達(dá)降低同時(shí)ASIC2a表達(dá)升高。這提示葡萄糖低代謝出現(xiàn)在癲癇大鼠自發(fā)性癲癇發(fā)作之前,可能發(fā)揮了促進(jìn)癲癇發(fā)生的作用。第二部分:糖缺乏的PC12細(xì)胞中TFCP2及ASIC2a的蛋白表達(dá)研究對(duì)PC12細(xì)胞進(jìn)行低糖培養(yǎng)基、無(wú)糖培養(yǎng)基、不同濃度1型葡萄糖轉(zhuǎn)運(yùn)體抑制劑SFT-31、不同培養(yǎng)環(huán)境下(高糖、無(wú)糖)不可代謝葡萄糖類似物2-DG處理后,利用蛋白免疫印跡技術(shù)檢測(cè)PC12細(xì)胞中TFCP2及ASIC2a的蛋白表達(dá)情況。結(jié)果顯示,低糖、無(wú)糖、STF-31、高糖培養(yǎng)條件下2-DG處理的PC12細(xì)胞中,TFCP2蛋白表達(dá)水平降低和ASIC2a蛋白表達(dá)水平升高。在無(wú)糖培養(yǎng)條件下,2-DG不引起TFCP2和ASIC2a的蛋白表達(dá)變化。這提示葡萄糖缺乏(細(xì)胞內(nèi)可利用糖或糖代謝中間產(chǎn)物的減少)能夠直接引起TFCP2和ASIC2a表達(dá)變化。第三部分:TFCP2和ASIC2a亞細(xì)胞表達(dá)定位及TFCP2對(duì)ASIC2a表達(dá)的調(diào)控作用研究通過(guò)免疫熒光標(biāo)記,對(duì)癲癇大鼠海馬區(qū)和糖缺乏的PC12細(xì)胞中的TFCP2和ASIC2a蛋白進(jìn)行定位。用TFCP2-siRNA和過(guò)表達(dá)TFCP2質(zhì)粒轉(zhuǎn)染PC12細(xì)胞,利用蛋白免疫印跡技術(shù)檢測(cè)PC12細(xì)胞中TFCP2及ASIC2a的蛋白表達(dá)情況。結(jié)果顯示,在大鼠CA1錐體神經(jīng)元和PC12細(xì)胞中ASIC2a主要表達(dá)于細(xì)胞膜和細(xì)胞質(zhì)中,而TFCP2主要表達(dá)于細(xì)胞質(zhì)和細(xì)胞核中。有趣的是,癲癇大鼠CA1錐體神經(jīng)元和糖缺乏PC12細(xì)胞中,位于細(xì)胞核內(nèi)TFCP2的熒光強(qiáng)度明顯減弱、甚至消失。PC12細(xì)胞中敲減TFCP2表達(dá)引起ASIC2a表達(dá)升高,而TFCP2過(guò)表達(dá)引起ASIC2a表達(dá)降低。這提示在整體細(xì)胞環(huán)境下TFCP2具有負(fù)向調(diào)控ASIC2表達(dá)的功能。第四部分:調(diào)控ASIC2a表達(dá)后影響神經(jīng)元內(nèi)源性興奮性和癲癇易感性的功能性研究利用陰性對(duì)照慢病毒、ASIC2a過(guò)表達(dá)慢病毒、ASIC2a敲減慢病毒感染器官特異型海馬腦片,通過(guò)膜片鉗系統(tǒng)檢測(cè)被病毒感染后的CA1椎體神經(jīng)元的內(nèi)源性興奮性。在大鼠CA1區(qū)通過(guò)腦離體定向注射陰性對(duì)照AAV、ASIC2a過(guò)表達(dá)AAV,待其穩(wěn)定表達(dá)后進(jìn)行匹魯卡品誘導(dǎo)的癲癇行為學(xué)檢測(cè)。結(jié)果顯示,在電生理試驗(yàn)中,ASIC2a過(guò)表達(dá)神經(jīng)元在相同電流強(qiáng)度刺激下誘發(fā)的動(dòng)作電位發(fā)放個(gè)數(shù)較陰性對(duì)照組增加;同時(shí)ASIC2a敲減神經(jīng)元在相同電流強(qiáng)度刺激下誘發(fā)的動(dòng)作電位發(fā)放個(gè)數(shù)較陰性對(duì)照組減少。ASIC2a表達(dá)模式改變后CA1椎體神經(jīng)元的細(xì)胞膜輸入阻抗無(wú)明顯變化。這提示ASIC2a過(guò)表達(dá)引起CA1錐體神經(jīng)元的內(nèi)源性興奮性升高,同時(shí)ASIC2a表達(dá)減少引起CA1錐體神經(jīng)元的內(nèi)源性興奮性降低。在行為學(xué)試驗(yàn)中,海馬區(qū)ASIC2a過(guò)表達(dá)組大鼠從注射匹魯卡品到Racine IV級(jí)的時(shí)間較對(duì)照組明顯減少,且達(dá)到Racine IV級(jí)的比例較對(duì)照組明顯增多。這提示CA1區(qū)ASIC2a過(guò)表達(dá)引起大鼠癲癇易感性的增加。我們的所有研究結(jié)果表明海馬區(qū)低代謝可以通過(guò)抑制TFCP2表達(dá)而誘導(dǎo)ASIC2a表達(dá)升高,繼而增強(qiáng)CA1錐體神經(jīng)元的內(nèi)源性興奮性,并最終導(dǎo)致增加顳葉癲癇的易感性。腦葡萄糖代謝不足可能是誘導(dǎo)癲癇發(fā)生的另一種可能的機(jī)制。這也為“癲癇發(fā)作誘導(dǎo)癲癇發(fā)作”假說(shuō)提供了可能的補(bǔ)充機(jī)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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R742.1
，
本文編號(hào)：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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R742.1
，
本文編號(hào)：2230101
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