血清腺苷酸酶活性与癫痫发作的相关性研究

发布时间：2018-04-12 16:53

本文选题：癫痫发作 + 三磷酸腺苷二磷酸水解酶　；参考：《郑州大学》2016年博士论文

【摘要】：研究背景与现状癫痫是神经系统较为常见且容易反复发作的疾病,癫痫致残率高、病程长,全世界癫痫患者数以千万,中国作为人口大国,癫痫患者有900多万,癫痫反复发作给患者及家庭造成极大的心理负担和经济负担。癫痫是以脑神经元过度放电所致的暂时性中枢神经功能紊乱为基本特征的脑组织慢性疾病,发作性、短暂性、重复性以及刻板性是其临床四大特点。近期,许多学者对癫痫病因、发病机制及病理学改变和治疗方法进行了研究并取得了较大进展。目前普遍认为,癫痫发生与突触重塑、神经递质受体、神经胶质细胞、离子通道、遗传因素、苔藓纤维发芽等多方面密切相关,痫性放电的开始伴有离子异常跨膜运动,通过突触联系和易化使放电逐渐传播,最终通过主动抑制和负反馈来终止痫性放电。但癫痫发作的的具体机制尚未明了,每次癫痫发作开始和终止的具体机制也未阐明。许多难治性癫痫患者应用多种抗癫痫药物并无显著效果,说明仍然需要寻找新的药理学作用靶点。因此,研究癫痫的发病机制、研究新的药理学作用靶点,对改善癫痫患者的预后及降低死亡率有重要的临床意义。许多相关研究表明,腺苷系统是脑组织中重要的抗癫痫和脑保护系统。其通过腺苷与G蛋白偶联腺苷受体相互作用来抑制脑组织内的神经元活性,急性癫痫发作后腺苷水平显著升高,随后发作终止,腺苷系统被称为“内源性抗癫痫系统”,可溶性核苷酸酶可与胞外核苷酸酶协同作用产生腺苷而在痫性发作中起着重要的调节作用。目前腺苷酸酶活性与癫痫的关系已引起广大医学工作者的重视。目前,癫痫发作后,患者血清ATP、ADP及AMP水解率改变以及血清可溶性磷酸二酯酶活性变化的相关报道较少;不同癫痫发作类型的血清ATP、ADP及AMP水解率改变以及血清可溶性磷酸二酯酶活性变化的是否相同未见有报道;腺苷A_1R与模型海马神经元损伤的关系方面的研究也较少。本文主要采用前瞻性研究纳入癫痫患者65例(全面性发作33例,部分性发作32例)和正常健康对照者35例,从第一部分和第二部分两个部分分别探讨了不同癫痫发作类型发作前后不同时间点血清ATP、ADP及AMP水解情况,不同癫痫发作类型发作前后不同时间点血清可溶性磷酸二酯酶活性变化情况,以了解腺苷酸酶活性与癫痫发作的关系以及不同癫痫发作类型腺苷酸酶活性有无差异,进一步探讨腺苷酸酶活性在癫痫发作过程中的作用机制,并深入了解不同癫痫发作类型对腺苷酸酶活性的敏感性,为临床选药提供依据;第三部分通过建立杏仁核点燃癫痫大鼠模型,从而对点燃癫痫大鼠各个时间段海马神经元的病理组织学和凋亡的影响进行分析,探讨腺苷A_1R与模型海马神经元损伤的关系,了解其对脑神经的保护作用,从而能更深刻的理解腺苷A_1R与癫痫间的关系,为癫痫的治疗提供参考。第一部分不同癫痫发作类型发作间期和癫痫发作后血清ATP、ADP和AMP水解率的动态变化目的:探讨患者发作间期和发作后血清ATP、ADP和AMP水解率的变化,分析ATP、ADP和AMP水解率与癫痫发作的关系,并分析不同癫痫发作类型癫痫发作后ATP、ADP和AMP水解率有无差异。方法:1.研究对象:收集2014年3月至2015年12月在我院神经内科治疗的癫痫患者65例,纳入病例组(全面性发作组33例,复杂部分性发作组32例)。病例组另外收集同期在我院门诊进行常规体检的正常人员35例作为健康对照组。2.观测指标:采用紫外线吸收分光光度法,观察病例组癫痫发作后5、10、20、30、60 min和10 h时的ATP、ADP和AMP水解率变化情况,与健康对照组比较;观察全面性发作组和复杂部分性发作组癫痫发作后5、10、20、30、60 min和10 h时的ATP、ADP和AMP水解率变化情况,并进行比较。3.统计学方法:采用SPSS21.0版本统计学软件进行数据处理,ATP或ADP或AMP水解率以(x±s)描述,采用重复测量数据的方差分析,组间比较采用t检验。结果:1.病例组患者在发作间期ATP水解率、ADP水解率、AMP水解率与对照组比较无统计学差异(P0.05);2.发作后5min时ATP水解率、ADP水解率、AMP水解率均明显升高,其中ATP水解率在发作后20 min达峰值,之后逐渐下降,至发作后10 h恢复至发作间期水平;ADP水解率、AMP水解率在发作后5 min达峰值,随后逐渐下降,至发作后60 min即恢复至发作间期水平(P0.05)。3.病例组发作后5min时患者血清ATP水解率、ADP水解率、AMP水解率均明显升高,病例组患者ATP水解率与对照组相比在发作后20 min达峰值,之后逐渐下降,至发作后10 h恢复至发作间期水平;ADP水解率、AMP水解率在发作后5 min达峰值,随后逐渐下降,至发作后60 min即恢复至发作间期水平(P0.05)。4.全面性发作组和部分性发作组相比,癫痫发作后5min,20 min血清ATP水解率、ADP水解率、AMP水解率升高更为显著(P0.05)。结论:癫痫发作后腺苷酸水解率明显升高,全面性发作患者与部分性发作患者相比,腺苷酸水解率升高的更为明显,提示腺苷酸酶活性增高可能参与了与癫痫发作终止相关的调节机制。第二部分不同癫痫发作类型血清可溶性磷酸二酯酶活性与癫痫发作的关系目的:探讨不同癫痫类型患者发作后血清可溶性磷酸二酯酶活性的变化情况,了解可溶性磷酸二酯酶活性在癫痫发作中的作用。方法:1.研究对象:收集2014年3月至2015年12月在我院神经内科治疗的癫痫患者65例,纳入病例组(全面性发作组33例,复杂部分性发作组32例)。同时收集常规体检的正常人员35例作为健康对照组。2.观测指标:采用紫外线吸收分光光度法,观察病例组患者发作后5min、10 min、20min和10 h时磷酸二酯酶水平、乳酸脱氢酶(LDH)水平变化情况并与正常对照组比较。观察全面性发作组和复杂部分性发作组癫痫发作后5min、10 min、20min和10 h时磷酸二酯酶水平、乳酸脱氢酶(LDH)水平变化,并进行比较。3.统计学方法:采用SPSS21.0版本统计学软件进行数据处理,磷酸二酯酶活性、LDH以(x±s)描述,采用重复测量数据的方差分析,组间比较采用t检验,以P0.05为差异有统计学意义。结果:1.病例组患者在发作间期磷酸二酯酶活性与对照组比较无统计学差异(P0.05)。2.病例组患者在发作后5min、10 min、20min时磷酸二酯酶活性明显升高,在发作后20 min时达峰值,之后逐渐下降,至发作后10 h恢复至发作间期水平(P0.05);3.全面性发作患者和部分性发作患者相比,发作后5min、10 min、20min时磷酸二酯酶活性明显升高,在发作后20 min时达峰值,之后逐渐下降,至发作后10 h恢复至发作间期水平(P0.05);4.LDH活性在发作间期、发作后不同时间点的变化均无统计学差异(P0.05)。结论:癫痫发作后磷酸二酯酶活性明显升高,全面性发作后磷酸二酯酶活性升高更为明显,磷酸二酯酶活性增高与ATP水解率密切相关,也进一步证明了癫痫发作后腺苷酸活性明显升高,这可能是癫痫发作终止的一个调节机制;全面性发作后磷酸二酯酶活性增高升高更为明显,证实腺苷酸活性对全面性发作的调节作用更强。第三部分腺苷A_1受体对杏仁核点燃癫痫大鼠海马神经元损伤作用机制目的:探讨腺苷A_1受体活性对点燃癫痫大鼠海马神经元损伤的作用,为癫痫发作的治疗提供理论依据。方法:1.研究对象:选择84只SPF级雄性Wistar大鼠,根据随机数字表法将动物分为正常组、致痫组、致痫+腺苷A_1R拮抗剂(8-环戊-1,3-二丙基黄嘌呤,DPCPX)组、致痫+腺苷A_1R激动剂(2-氯化腺苷,2-CADO)4个分组,每组21只。致痫组、致痫+DPCPX组、致痫+2-CADO组采用电刺激点燃癫痫模型。正常组未进行手术和电刺激致痫等处理;致痫组癫痫模型建立成功前后未注射任何药物;致痫+DPCPX组大鼠癫痫模型建立成功前1h和成功后1 h尾部静脉注射0.3mg/kg DPCPX;致痫+2-CADO组大鼠癫痫模型成功前1h和成功后1 h尾部静脉注射0.6mg/kg 2-CADO。2.观测指标:采用TUNEL法,观察各组在癫痫模型建立成功后1天、15天、30天时的海马CA3区组织学病理变化及神经元凋亡指数(AI)变化情况。3.统计学方法:符合正态分布数据用(x±s)描述,采用t检验,以P0.05为差异有显著性,统计学软件选用SPSS21.0版本。结果:1.致痫组、致痫+DPCPX组、致痫+2-CADO组动物在点燃癫痫后1天、15天、30天均出现不同程度的神经元细胞排列松散无规则,轮廓模糊,边缘欠清晰,胞核萎缩,胞浆空泡等神经元结构损害;2.相同时间点,致痫+DPCPX组的神经元结构损害程度较致痫组加重,致痫+2-CADO组较致痫组减轻。正常组在不同时间点的AI变化不明显(P0.05),致痫组、致痫+DPCPX组、致痫+2-CADO组的AI随着癫痫发作时间的延长,AI逐渐增加(P0.05);3.在相同时间点,致痫组、致痫+DPCPX组、致痫+2-CADO组的AI均明显高于正常组(P0.05),致痫+DPCPX组的AI均明显高于致痫组(P0.05);致痫+2-CADO组的AI明显低于致痫组和致痫+DPCPX组(P0.05)。结论:癫痫发作会引起神经元损伤和凋亡,腺苷A_1R活性降低,神经元损伤和凋亡更为明显,这可能是癫痫发作后神经元损伤的机制之一;通过增加腺苷A_1R活性可保护神经元,降低其损伤,可能是终止癫痫发作的一个治疗方向。
[Abstract]:The research background and current situation of epilepsy is the nervous system is common and easy to recurrent disease, epilepsy, high disability rate, long duration of the disease, the world tens of million of patients with epilepsy, Chinese as a country with a large population, there are about 9000000 patients with epilepsy, seizures caused great psychological burden and economic burden to patients and families with epilepsy is brain. Neurons over temporary central nerve dysfunction caused by the discharge of the basic features of the brain tissue of chronic disease, recurrent, transient, repetitive and stereotyped is four of its clinical characteristics. Recently, many scholars of epilepsy etiology, studied the change and treatment of pathogenesis and pathological methods and made great progress. It is generally believed that synaptic remodeling and epileptogenesis, neurotransmitter receptors, glial cells, ion channels, genetic factors, mossy fiber sprouting and other aspects are closely related, epileptic discharge Electric started with abnormal transmembrane ion movement through synaptic connections and easy to discharge gradually spread, through active suppression and negative feedback to the termination of epileptiform discharge. But the specific mechanism of epileptic seizures is not clear, each specific mechanism of start and end is not clear. Many patients with intractable epilepsy a variety of anti epilepsy drugs had no significant effect, still need to find new targets for pharmacological effects. Therefore, studying the mechanism of epilepsy, pharmacological research of new targets, to improve the prognosis of patients with epilepsy and reduce the death rate has important clinical significance. Many studies indicate that adenosine is anti epilepsy and brain system the important protection systems in the brain. By adenosine and G protein coupled adenosine receptor interaction to inhibition of neuronal activity in the brain tissue of acute seizures after adenosine levels significantly Increased, then werecured, adenosine system known as the "endogenous antiepileptic system", soluble extracellular nucleotides and nucleotide enzyme enzymes produce adenosine in epileptic plays an important regulatory role. At present the relationship between ATPase activities and epilepsy has aroused the attention of medical workers. At present, seizure after the serum ATP, ADP and AMP hydrolysis rate changes and serum soluble phosphodiesterase activity changes in the relevant reports; different seizure types of serum ATP, ADP and AMP hydrolysis rate changes and soluble phosphodiesterase activity changes are the same has not been reported; less research on the relationship between adenosine A_1R and model of hippocampal neurons induced by hand. This paper mainly uses the prospective study included 65 patients with epilepsy (GTCS in 33 cases, partial seizures in 32 cases) and healthy controls were 3 In 5 cases, from two parts: the first part and the second part respectively discusses the types of attacks at different times after serum ATP different seizures, ADP and AMP hydrolysis, different types of seizure onset at different time points before and after the changes of serum soluble phosphodiesterase activity, to explore the relationship between ATPase activities and epileptic seizures and seizure type there is no difference in ATPase activities, to further explore the ATPase activities in the mechanism in the process of epilepsy, and deeply understand the type of ATPase activities sensitivity of different seizures, provide the basis for clinical drug selection; the third part through the establishment of model of amygdala in epileptic rats kindled, analyze and influence lit each time epilepsy rat hippocampal neurons apoptosis and histopathology, adenosine A_1R and model of hippocampal neurons shut The Department, to understand its protective effect on brain nerve, which can be more profound understanding of the relationship between adenosine A_1R and epilepsy, and provide reference for the treatment of epilepsy. The first part of different types of seizure and interictal seizures after serum ATP, the dynamic changes of ADP and the hydrolysis rate of AMP Objective: to investigate the patients with interictal and after seizure serum ATP, ADP and AMP hydrolysis rate changes, analysis of ATP, ADP and AMP and the relationship between the hydrolysis rate of seizures, and analyze the types of epilepsy after ATP seizures, ADP and AMP have no difference in the hydrolysis rate. Methods: 1. subjects: from March 2014 to December 2015 in our hospital treatment of epilepsy patients in 65 cases, including cases (general seizure group 33 cases, 32 cases of complex partial seizures group). Normal group were also collected from the personnel routine health examination in our hospital 35 cases as healthy control group.2. observation index: The ultraviolet absorption spectrophotometry, observe cases after the seizure of 5,10,20,30,60 min and 10 h ATP, ADP and AMP hydrolysis rate changes, compared with the healthy control group; observation group and general seizure patients with complex partial seizure epilepsy after 5,10,20,30,60 min and 10 h ATP, ADP and AMP hydrolysis the rate of change, and compare the.3. statistical method: the data were processed by statistical software SPSS21.0 version, ATP or ADP or AMP on the hydrolysis rate (x + s), using the analysis of variance of repeated measurement data, comparison between groups with t test. Results: 1. cases of patients in the interictal ATP hydrolysis rate. ADP the rate of hydrolysis, the hydrolysis rate of AMP and the control group showed no significant difference (P0.05); 2. 5min after the onset of ATP hydrolysis rate, ADP rate of hydrolysis, the hydrolysis rate of AMP were significantly increased, the hydrolysis rate of ATP after the onset of 20 min reached the peak, then gradually declined to attack After 10 h recovery to interictal levels; the rate of ADP hydrolysis, the hydrolysis rate of AMP after the onset of 5 min reached the peak, then gradually decreased to 60 min after the onset of recovery to interictal levels (P0.05) in patients with.3. after the onset of 5min in serum of patients with ATP the rate of hydrolysis, the hydrolysis rate of ADP, the hydrolysis rate was obviously AMP increased patients ATP hydrolysis rate compared with the control group after the onset of 20 min reached the peak, then gradually declined to 10 h after the onset of recovery to interictal levels; the rate of ADP hydrolysis, the hydrolysis rate of AMP after the onset of 5 min reached the peak, then gradually decreased to 60 min after the onset of recovery to interictal the level of (P0.05).4. general seizure group and partial seizure group compared to 5min after seizure, 20 min serum ATP hydrolysis rate, ADP rate of hydrolysis, the hydrolysis rate of AMP were higher (P0.05). Conclusion: after the seizure of adenylate hydrolysis rate increased significantly, generalized seizures and partial Patients compared to adenylate hydrolysis rate was further up-regulated, suggesting ATPase activity increased may be involved in the termination of regulatory mechanisms associated with seizures. Second different seizure types of relationship between serum soluble phosphodiesterase activity and epilepsy Objective: To investigate the changes of different types of epilepsy patients after the onset of serum soluble phosphodiesterase activity, role understanding the soluble phosphodiesterase activity in epilepsy. Methods: 1. subjects: from March 2014 to December 2015 in our hospital treatment of patients with epilepsy in 65 cases, including cases (general seizure group 33 cases, complex partial seizures group 32 cases). The normal staff also collected the routine physical examination in 35 cases as healthy control group..2. measurements: using ultraviolet absorption spectrophotometry, observation of patients after the onset of 5min, 10 20min and 10 min. H phosphodiesterase levels of lactate dehydrogenase (LDH) levels were compared with the normal control group. The observation group comprehensive seizures and complex partial seizures of epilepsy after 5min group, 10 min 20min and 10 h phosphodiesterase levels, lactate dehydrogenase (LDH) levels, and compare the.3. statistical method: data using SPSS21.0 statistical software, version LDH with phosphodiesterase activity, (x + s), using the analysis of variance of repeated measurement data, were compared by t test, with P0.05 as the difference was statistically significant. Results: 1. cases of patients in the interictal phosphodiesterase activity compared with the control group no significant difference (P0.05).2. patients after the onset of 5min, 10 min, 20min phosphodiesterase activity was significantly increased, and reached a peak in the attack after 20 min, then gradually declined to 10 h after the onset of recovery to interictal water Ping (P0.05); compared with 3. generalized seizures and partial seizures in patients after the onset of 5min, 10 min, 20min phosphodiesterase activity was significantly increased, and reached a peak in the attack after 20 min, then gradually declined to 10 h after the onset of recovery to interictal levels (P0.05); the activity of 4.LDH in interictal. After the onset of changes at different time points were not statistically significant (P0.05). Conclusion: phosphodiesterase activity increased significantly after seizures, generalized seizures after phosphodiesterase activity increased more significantly, increased phosphodiesterase activity is closely related with the hydrolysis rate of ATP, is further evidence that adenosine activity increased significantly after seizures, which may be a regulation the mechanism of seizure termination; general seizure after increased phosphodiesterase activity increased more significantly, with stronger activity of adenylate confirmed on the regulation of general seizure. In the third part, adenosine A_1 Receptor on amygdala kindling injury of hippocampal neurons in epileptic rats Objective: To investigate the mechanism of adenosine A_1 receptor activity of hippocampal neurons in epileptic rats kindled injury, and provide a theoretical basis for the treatment of epilepsy. Methods: 1. subjects: 84 male Wistar SPF rats, then randomly divided into animal normal group, epilepsy group, epilepsy induced by adenosine A_1R antagonist (8- + cyclopent -1,3- two propyl xanthine, DPCPX) group, epileptic (2- + adenosine A_1R agonist adenosine chloride, 2-CADO) 4 groups, 21 rats in each group. Epileptic seizure group + DPCPX group, +2-CADO group with epilepsy electrical kindling model of epilepsy. The normal group did not undergo surgery and electrical stimulation treatment of epileptic seizure groups; epilepsy model was established successfully without any medication before and after injection; epileptic +DPCPX rats epileptic model was established successfully 1H before and after the success of 1 h tail intravenous injection of 0.3mg/kg DPCPX; epilepsy 1 h tail intravenous injection of 0.6mg/kg 2-CADO.2. observed in +2-CADO rats model of epilepsy before and after the success of the success of 1H: the TUNEL method was observed in the epilepsy model was established successfully after 1 days, 15 days, pathological changes and neuronal apoptosis index at 30 days in CA3 area of hippocampus tissue (AI) changes of.3. statistical methods: accord with normal distribution data (x + s), using t test, P0.05 with significant difference, with statistical software SPSS21.0 version. Results: 1. epileptic seizure group, +DPCPX group, +2-CADO group of epileptic animal in kindled seizures after 1 days, 15 days, 30 days there are neurons different degrees of loosely arranged irregular contour, fuzzy edge, less clear, nucleus atrophy, cytoplasmic vacuoles and neuron damage; 2. at the same time, the degree of neuronal damage in +DPCPX group compared with the structure of epileptic seizure groups increased, epileptic + 2-CADO group than in normal group. Epileptic No obvious changes in AI group at different time points (P0.05), epilepsy group, epilepsy group +DPCPX, epilepsy group +2-CADO AI with prolonged seizures, AI gradually increased (P0.05); 3. at the same time, epilepsy group, epilepsy group +DPCPX, epilepsy group +2-CADO AI were significantly higher than normal group (P0.05), epilepsy group +DPCPX AI were significantly higher than that of epilepsy group (P0.05); epilepsy +2-CADO group AI was significantly lower than that of epilepsy group and epilepsy group +DPCPX (P0.05). Conclusion: seizures can cause neuronal injury and apoptosis, reduce the activity of adenosine A_1R injury and apoptosis. Neuronal loss is more obvious, which may be one of the mechanisms of neuronal damage after seizures; by increasing the activity of adenosine A_1R can protect neurons, reduce the damage, may be the termination of a treatment of epilepsy.

【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R742.1

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