Dynamin-1與內(nèi)側(cè)顳葉癲癇發(fā)生的關系及機制研究
發(fā)布時間：2018-07-27 13:03
【摘要】：研究背景與目的： 癲癇(epilepsy)是一種常見的神經(jīng)系統(tǒng)疾病,在兒童期發(fā)病率明顯高于其他年齡階段,發(fā)病率約為3.5‰-6.6‰,其發(fā)病形式多樣,病因復雜,對病人的危害巨大。其中難治性癲癇以內(nèi)側(cè)顳葉癲癇(mesial temporal lobe epilepsy, MTLE)較為常見,海馬硬化為其突出的臨床病理特征。迄今為止,MTLE的治療一直是癲癇治療的難點,MTLE的發(fā)病機制仍是近幾十年來癲癇研究的熱點與難點。 近幾年來,關于突觸囊泡循環(huán)相關調(diào)控蛋白的研究已成為一個新的熱點。突觸囊泡循環(huán)是維持神經(jīng)遞質(zhì)傳遞的基礎,突觸囊泡通過胞吐和胞吞作用完成一次突觸囊泡循環(huán)。大量研究證實,癲癇患者神經(jīng)元突觸有明顯的功能異常,這種病態(tài)突觸通過突觸囊泡的快速循環(huán)再生使正常情況下每秒僅能傳播數(shù)次或數(shù)十次神經(jīng)沖動的突觸傳遞功能增加到每秒數(shù)十次到數(shù)百次,使癇性放電得以迅速擴布。深入研究囊泡的再循環(huán)細節(jié)以及生理調(diào)控的方式和位點,為臨床尋找有效治療靶點,具有重要的理論和實際意義。 Dynamin-1,又稱發(fā)動蛋白-1,是一個96kD的大分子量三磷酸鳥苷酶(GTPase),為GTPase家族成員,近20年的研究從基因、細胞信號調(diào)控、蛋白功能等多方面已證實,dynamin-1在網(wǎng)格蛋白介導的突觸囊泡胞吞及囊泡循環(huán)利用中發(fā)揮至關重要的作用。非dynamin-1依賴的其他調(diào)控機制能夠維持生理狀態(tài)下基本正常的突觸囊泡胞吞功能,而dynamin-1在維持神經(jīng)元高頻、長時程放電過程中發(fā)揮了關鍵作用。因此,以dynamin-1作為靶點進行干預,可以阻斷囊泡在高頻、長時程電興奮事件中的快速循環(huán)而發(fā)揮作用,而對生理狀態(tài)下的突觸傳遞活動影響甚少。由此可見,dynamin-1有希望成為一個非常有前景、完全不同于傳統(tǒng)抗癲癇藥物作用機制的抗癲癇新藥研發(fā)靶點。 本實驗通過建立幼年大鼠MTLE模型,從一個整體、連續(xù)發(fā)展的角度探討dynamin-1在MTLE發(fā)生發(fā)展過程中的表達變化,同時在人MTLE患兒海馬組織中進行驗證。并進一步建立離體原代海馬神經(jīng)元癲癇細胞模型,探討dynamin-1在癲癇發(fā)生過程中的機制。從而為難治性MTLE尋找新的藥物靶點提供了一個全新的視角。 隨后,本實驗通過構(gòu)建dynamin-14個結(jié)構(gòu)域的融合蛋白,在大鼠腦神經(jīng)突觸小體中篩選出63個可能與dynamin-1存在相互作用的蛋白,并選取了3個以前從未報道過與dynamin-1有相互作用的蛋白：Rab GDⅠ、C1C-3以TUC-4b,利用免疫共沉淀對其進行自然條件下與dynamin-1相互作用的研究。 本研究分為三部分： 研究方法： 1.利用本實驗室已建立的方法,用氯化鋰-匹羅卡品誘導3周齡Sprague-Dawley (SD)幼鼠出現(xiàn)癲癇發(fā)作,根據(jù)MTLE發(fā)生發(fā)展過程取三個時間點(急性期、潛伏期、慢性期)一共分為6個組：急性期對照組、急性期模型組、潛伏期對照組、潛伏期模型組、慢性期自發(fā)發(fā)作組、慢性期對照組。運用western blot及免疫組化技術動態(tài)觀察MTLE大鼠海馬組織中dynamin-1及磷酸化dynamin-1蛋白的表達變化。并收集人難治性MTLE術后海馬標本,western blot及免疫組化方法檢測其表達變化。 2.利用新生SD大鼠培養(yǎng)海馬神經(jīng)元,應用無鎂細胞外液灌流方法誘導癇性放電,建立癲癇細胞模型。應用dynamin-1抑制劑dynasore抑制dynamin-1的活性,western blot及免疫熒光技術觀察癲癇細胞模型中dynamin-1及磷酸化dynamin-1蛋白的表達變化,并利用轉(zhuǎn)鐵蛋白進一步研究抑制dynamin-1蛋白活性后癲癇細胞模型胞吞量的改變。 3.構(gòu)建dynamin-14個功能結(jié)構(gòu)域的融合蛋白,與大鼠腦突觸小體相互作用,利用GST-pull down聯(lián)合質(zhì)譜技術篩選出dynamin-1在大鼠腦神經(jīng)突觸小體中的相互作用蛋白。驗證2個已有報道中確切與dynamin-1存在相互作用的蛋白,endophilins以及amphiphysins。并選取3個以前從未報道過的dynamin-1相互作用的蛋白,Rab GDⅠ、C1C-3以及TUC-4b,應用免疫共沉淀對其進行自然條件下與dynamin-1相互作用的研究。 研究結(jié)果： 1. Western blot及免疫組化結(jié)果顯示磷酸化dynamin-1蛋白在MTLE大鼠急性期及慢性期以及MTLE患兒海馬組織中表達較同時期對照組顯著降低(P0.05),在MTLE大鼠潛伏期的表達與同時期對照組相比無差異。而dynamin-1總蛋白在MTLE大鼠各期、MTLE患兒及對照組海馬之間表達無明顯差異。 2. Western blot及免疫組化結(jié)果顯示磷酸化dynamin-1蛋白在癲癇細胞模型組中表達較對照組顯著降低(P0.05),經(jīng)dynamin-1抑制劑dynasore預處理的癲癇細胞模型組中磷酸化dynamin-1蛋白表達則與正常對照組基本一致,同時,dynasore對正常對照組磷酸化dynamin-1蛋白表達無明顯影響。Dynamin-1總蛋白在海馬神經(jīng)元癇性放電模型及對照組中表達無明顯差異。激光顯微共聚焦顯示癲癇細胞模型組海馬神經(jīng)元胞吞量較正常對照組明顯增多(P0.05)。應用dynasore抑制dynamin-1蛋白活性表達后,癇性放電組海馬神經(jīng)元胞吞量顯著減少。同時,dynasore對正常對照組海馬神經(jīng)元胞吞量無明顯影響。 3.本實驗共篩選出63個可能與dynamin-1存在相互作用的蛋白,其中PRD域鑒定出36個蛋白,PH域14個蛋白,GED域7個蛋白,GTPase域6個蛋白。已知的dynamin-1相互作用蛋白endophilins和amphiphysins被證實與dynamin-1存在相互作用關系。未報道蛋白RabGDI以及C1C-3與dynamin-1存在相互作用關系,然而,TUC-4b與dynamin-1之間未檢測出相互作用。 研究結(jié)論： 1.磷酸化dynamin-1蛋白在MTLE大鼠急性期、慢性期以及MTLE患兒海馬組織中表達較同時期對照組顯著降低,提示dynamin-1可能通過磷酸化／去磷酸化過程在癲癇發(fā)生發(fā)展過程中發(fā)揮重要作用。 2.癲癇細胞模型組磷酸化dynamin-1蛋白表達下降,轉(zhuǎn)鐵蛋白胞吞量增多,但抑制dynamin-1活性后,其表達與正常對照組無明顯變化,同時正常對照組dynamin-1活性抑制前后磷酸化dynamin-1蛋白表達無明顯差異。提示dynamin-1可能通過影響其磷酸化／去磷酸化及調(diào)節(jié)突觸囊泡胞吞在癲癇發(fā)病機制中起重要作用,但對生理狀態(tài)下正常細胞表達無影響。 3.篩選了63個可能與dynamin-1存在相互作用的蛋白。證實endophilins和amphiphysins蛋白與dynamin-1存在相互作用關系。首次報道蛋白Rab GDI以及C1C-3與dynamin-1作用相關。
[Abstract]:Research background and purpose:
Epilepsy (epilepsy) is a common nervous system disease. The incidence of epilepsy in childhood is obviously higher than that of other ages. The incidence of epilepsy is about 3.5 per thousand -6.6 per thousand. The incidence of epilepsy is varied, the cause is complex and the disease is very harmful to the patients. Among them, the intractable epilepsy (mesial temporal lobe epilepsy, MTLE) is more common, and the hippocampus is sclerosis. So far, the treatment of MTLE has been a difficult point for the treatment of epilepsy, and the pathogenesis of MTLE is still a hot and difficult point in recent decades.
In recent years, the study of synaptic vesicle cycle related regulatory proteins has become a new hot spot. Synaptic vesicle circulation is the basis for maintaining neurotransmitter transmission. Synaptic vesicles complete a synaptic vesicle cycle through exocytosis and endocytosis. A large number of studies have proved that the neuronal synapses in epileptic patients have obvious functional abnormalities. The rapid circulatory regeneration of synapses through synaptic vesicles makes synaptic transmission functions that can only transmit several or dozens of nerve impulses per second to hundreds of times per second to hundreds of times per second in normal conditions, making the epileptic discharge rapidly spread. Effective therapeutic targets have important theoretical and practical significance.
Dynamin-1, also known as the starting protein -1, is a 96kD macromolecular weight three guanosine enzyme (GTPase), a member of the GTPase family. In the past 20 years, the research has been confirmed in many aspects, such as gene, cell signal regulation, protein function, and so on. Dynamin-1 plays a vital role in the use of grid protein mediated synaptic vesicle endocytosis and vesicle recycling. Other regulatory mechanisms of IN-1 dependence can maintain the basic normal synaptic vesicle function under physiological state, and dynamin-1 plays a key role in maintaining the high frequency and long term discharge process of neurons. Therefore, the intervention of dynamin-1 as a target can block the rapid circulation of vesicles in high frequency and long term electrical excitatory events. It can be seen that dynamin-1 is expected to be a very promising, completely different from the traditional antiepileptic drug mechanism of antiepileptic drugs research and development target.
In this experiment, the MTLE model of young rats was established to explore the expression changes of dynamin-1 during the development of MTLE from a whole and continuous development point of view. At the same time, it was verified in the hippocampal tissue of children with MTLE, and the epileptic cell model of the isolated primary hippocampal neurons was further established to explore the mechanism of dynamin-1 in the process of epilepsy. This provides a new perspective for the search of new drug targets for refractory MTLE.
Then, we screened 63 proteins that might interact with dynamin-1 in the synaptic body of the rat brain by constructing the fusion protein of dynamin-14 domain, and selected 3 proteins that had never been interacted with dynamin-1 previously reported: Rab GD I, C1C-3 with TUC-4b, and use immunoprecipitation for its nature. The study of the interaction with dynamin-1 under conditions.
This study is divided into three parts:
Research methods:
1. using the method established in this laboratory, lithium chloride pilocarpine was used to induce epileptic seizures in young rats of 3 weeks old Sprague-Dawley (SD). According to the development process of MTLE, three time points (acute, latent, chronic) were divided into 6 groups: acute phase control group, acute stage model group, latent period control group, latent period model group, slow The changes in the expression of dynamin-1 and phosphorylated dynamin-1 protein in the hippocampus of MTLE rats were dynamically observed by Western blot and immunohistochemical technique. The expressions of the hippocampal dynamin-1 after intractable MTLE were collected, and the expression changes were detected by Western blot and immunohistochemistry.
2. the hippocampal neurons were cultured in the newborn SD rats. The epileptic discharge was induced by the method of MGC external fluid perfusion. The epileptic cell model was established. The activity of dynamin-1 was inhibited by the dynamin-1 inhibitor dynasore. The expression of dynamin-1 and phosphorylated dynamin-1 protein in the epileptic cell model was observed by Western blot and immunofluorescence. Further studies on the changes of endocytosis in the epileptic cell model after inhibiting the activity of dynamin-1 protein were carried out with transferrin.
3. a fusion protein of dynamin-14 functional domains was constructed and interacted with the synaptosomes in the rat brain. The interaction proteins of dynamin-1 in the synaptic body of the rat brain were screened by GST-pull down combined mass spectrometry. The protein, endophilins and amphiphysins that had been confirmed to be exactly interacting with dynamin-1 were reported. 3 proteins interacting with dynamin-1, Rab GD I, C1C-3 and TUC-4b, which have never been reported before, were selected to study the interaction with dynamin-1 in natural conditions by immunoprecipitation.
The results of the study:
1. Western blot and immunohistochemical results showed that the expression of phosphorylated dynamin-1 protein in the acute and chronic phase of MTLE rats and in the hippocampus of MTLE children was significantly lower than that of the same period control group (P0.05). The expression of the latent period in the MTLE rats was no difference compared with the same period control group, but the total dynamin-1 protein was in the MTLE rat and MTLE children. There was no significant difference in the expression of the hippocampus between the control group and the control group.
2. Western blot and immunohistochemical results showed that the expression of phosphorylated dynamin-1 protein in the epileptic cell model group was significantly lower than that in the control group (P0.05). The expression of phosphorylated dynamin-1 protein in the epileptic cell model group pretreated by dynamin-1 inhibitor dynasore was basically consistent with that of the normal control group, and dynasore against the normal control group. The expression of dynamin-1 protein had no obvious effect on the expression of.Dynamin-1 total protein in the epileptic discharge model of hippocampal neurons and in the control group. Laser microconfocal microscopy showed that the amount of hippocampal neurons in the epileptic cell model group increased significantly (P0.05). After the application of dynasore to inhibit the expression of dynamin-1 protein, the epileptogenic activity was observed. The amount of cytosolic carbon in hippocampal neurons of the discharge group was significantly reduced. Meanwhile, dynasore had no significant effect on the amount of neurons in the hippocampus of the normal control group.
3. we screened 63 proteins that might interact with dynamin-1, in which 36 proteins were identified in the PRD domain, 14 in PH domain, 7 in the GED domain and 6 in the GTPase domain. The known dynamin-1 interacting protein endophilins and amphiphysins were confirmed to interact with dynamin-1. No protein RabGDI and C1C were reported. There is interaction between -3 and dynamin-1. However, no interaction was found between TUC-4b and dynamin-1.
The conclusions are as follows:
The expression of 1. phosphorylated dynamin-1 protein in the acute, chronic, and hippocampal tissues of MTLE rats was significantly lower than that of the same period control group, suggesting that dynamin-1 may play an important role in the development of epilepsy through the process of phosphorylation / dephosphorylation.
2. the expression of phosphorylated dynamin-1 protein in the epileptic cell model group decreased and the amount of transferrin swallowed increased, but the expression of the dynamin-1 activity was not significantly changed after the inhibition of the activity of the normal control group, and there was no significant difference in the expression of phosphorylated dynamin-1 protein before and after the inhibition of dynamin-1 activity in the normal control group. It suggested that dynamin-1 may affect its phosphorylation. Dephosphorylation and regulation of synaptic vesicle endocytosis play an important role in the pathogenesis of epilepsy, but have no effect on normal cell expression in physiological condition.
3. 63 proteins that might interact with dynamin-1 were screened. It was confirmed that endophilins and amphiphysins proteins interact with dynamin-1. The first reports of protein Rab GDI and C1C-3 are related to the action of dynamin-1.
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R742.1
[Abstract]:Research background and purpose:
Epilepsy (epilepsy) is a common nervous system disease. The incidence of epilepsy in childhood is obviously higher than that of other ages. The incidence of epilepsy is about 3.5 per thousand -6.6 per thousand. The incidence of epilepsy is varied, the cause is complex and the disease is very harmful to the patients. Among them, the intractable epilepsy (mesial temporal lobe epilepsy, MTLE) is more common, and the hippocampus is sclerosis. So far, the treatment of MTLE has been a difficult point for the treatment of epilepsy, and the pathogenesis of MTLE is still a hot and difficult point in recent decades.
In recent years, the study of synaptic vesicle cycle related regulatory proteins has become a new hot spot. Synaptic vesicle circulation is the basis for maintaining neurotransmitter transmission. Synaptic vesicles complete a synaptic vesicle cycle through exocytosis and endocytosis. A large number of studies have proved that the neuronal synapses in epileptic patients have obvious functional abnormalities. The rapid circulatory regeneration of synapses through synaptic vesicles makes synaptic transmission functions that can only transmit several or dozens of nerve impulses per second to hundreds of times per second to hundreds of times per second in normal conditions, making the epileptic discharge rapidly spread. Effective therapeutic targets have important theoretical and practical significance.
Dynamin-1, also known as the starting protein -1, is a 96kD macromolecular weight three guanosine enzyme (GTPase), a member of the GTPase family. In the past 20 years, the research has been confirmed in many aspects, such as gene, cell signal regulation, protein function, and so on. Dynamin-1 plays a vital role in the use of grid protein mediated synaptic vesicle endocytosis and vesicle recycling. Other regulatory mechanisms of IN-1 dependence can maintain the basic normal synaptic vesicle function under physiological state, and dynamin-1 plays a key role in maintaining the high frequency and long term discharge process of neurons. Therefore, the intervention of dynamin-1 as a target can block the rapid circulation of vesicles in high frequency and long term electrical excitatory events. It can be seen that dynamin-1 is expected to be a very promising, completely different from the traditional antiepileptic drug mechanism of antiepileptic drugs research and development target.
In this experiment, the MTLE model of young rats was established to explore the expression changes of dynamin-1 during the development of MTLE from a whole and continuous development point of view. At the same time, it was verified in the hippocampal tissue of children with MTLE, and the epileptic cell model of the isolated primary hippocampal neurons was further established to explore the mechanism of dynamin-1 in the process of epilepsy. This provides a new perspective for the search of new drug targets for refractory MTLE.
Then, we screened 63 proteins that might interact with dynamin-1 in the synaptic body of the rat brain by constructing the fusion protein of dynamin-14 domain, and selected 3 proteins that had never been interacted with dynamin-1 previously reported: Rab GD I, C1C-3 with TUC-4b, and use immunoprecipitation for its nature. The study of the interaction with dynamin-1 under conditions.
This study is divided into three parts:
Research methods:
1. using the method established in this laboratory, lithium chloride pilocarpine was used to induce epileptic seizures in young rats of 3 weeks old Sprague-Dawley (SD). According to the development process of MTLE, three time points (acute, latent, chronic) were divided into 6 groups: acute phase control group, acute stage model group, latent period control group, latent period model group, slow The changes in the expression of dynamin-1 and phosphorylated dynamin-1 protein in the hippocampus of MTLE rats were dynamically observed by Western blot and immunohistochemical technique. The expressions of the hippocampal dynamin-1 after intractable MTLE were collected, and the expression changes were detected by Western blot and immunohistochemistry.
2. the hippocampal neurons were cultured in the newborn SD rats. The epileptic discharge was induced by the method of MGC external fluid perfusion. The epileptic cell model was established. The activity of dynamin-1 was inhibited by the dynamin-1 inhibitor dynasore. The expression of dynamin-1 and phosphorylated dynamin-1 protein in the epileptic cell model was observed by Western blot and immunofluorescence. Further studies on the changes of endocytosis in the epileptic cell model after inhibiting the activity of dynamin-1 protein were carried out with transferrin.
3. a fusion protein of dynamin-14 functional domains was constructed and interacted with the synaptosomes in the rat brain. The interaction proteins of dynamin-1 in the synaptic body of the rat brain were screened by GST-pull down combined mass spectrometry. The protein, endophilins and amphiphysins that had been confirmed to be exactly interacting with dynamin-1 were reported. 3 proteins interacting with dynamin-1, Rab GD I, C1C-3 and TUC-4b, which have never been reported before, were selected to study the interaction with dynamin-1 in natural conditions by immunoprecipitation.
The results of the study:
1. Western blot and immunohistochemical results showed that the expression of phosphorylated dynamin-1 protein in the acute and chronic phase of MTLE rats and in the hippocampus of MTLE children was significantly lower than that of the same period control group (P0.05). The expression of the latent period in the MTLE rats was no difference compared with the same period control group, but the total dynamin-1 protein was in the MTLE rat and MTLE children. There was no significant difference in the expression of the hippocampus between the control group and the control group.
2. Western blot and immunohistochemical results showed that the expression of phosphorylated dynamin-1 protein in the epileptic cell model group was significantly lower than that in the control group (P0.05). The expression of phosphorylated dynamin-1 protein in the epileptic cell model group pretreated by dynamin-1 inhibitor dynasore was basically consistent with that of the normal control group, and dynasore against the normal control group. The expression of dynamin-1 protein had no obvious effect on the expression of.Dynamin-1 total protein in the epileptic discharge model of hippocampal neurons and in the control group. Laser microconfocal microscopy showed that the amount of hippocampal neurons in the epileptic cell model group increased significantly (P0.05). After the application of dynasore to inhibit the expression of dynamin-1 protein, the epileptogenic activity was observed. The amount of cytosolic carbon in hippocampal neurons of the discharge group was significantly reduced. Meanwhile, dynasore had no significant effect on the amount of neurons in the hippocampus of the normal control group.
3. we screened 63 proteins that might interact with dynamin-1, in which 36 proteins were identified in the PRD domain, 14 in PH domain, 7 in the GED domain and 6 in the GTPase domain. The known dynamin-1 interacting protein endophilins and amphiphysins were confirmed to interact with dynamin-1. No protein RabGDI and C1C were reported. There is interaction between -3 and dynamin-1. However, no interaction was found between TUC-4b and dynamin-1.
The conclusions are as follows:
The expression of 1. phosphorylated dynamin-1 protein in the acute, chronic, and hippocampal tissues of MTLE rats was significantly lower than that of the same period control group, suggesting that dynamin-1 may play an important role in the development of epilepsy through the process of phosphorylation / dephosphorylation.
2. the expression of phosphorylated dynamin-1 protein in the epileptic cell model group decreased and the amount of transferrin swallowed increased, but the expression of the dynamin-1 activity was not significantly changed after the inhibition of the activity of the normal control group, and there was no significant difference in the expression of phosphorylated dynamin-1 protein before and after the inhibition of dynamin-1 activity in the normal control group. It suggested that dynamin-1 may affect its phosphorylation. Dephosphorylation and regulation of synaptic vesicle endocytosis play an important role in the pathogenesis of epilepsy, but have no effect on normal cell expression in physiological condition.
3. 63 proteins that might interact with dynamin-1 were screened. It was confirmed that endophilins and amphiphysins proteins interact with dynamin-1. The first reports of protein Rab GDI and C1C-3 are related to the action of dynamin-1.
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R742.1
【共引文獻】
相關期刊論文 前10條
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