2013-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/650163摘要：癲癇會造成海馬迴的神經興奮性增加，神經細胞的新生以及在海馬齒狀回(dentategyrus)中的顆粒細胞(granulecell)向hilus的異位移動，其機轉尚未明瞭。SDF-1是一個小分子蛋白，在腦中具有刺激神經前驅幹細胞(neuronprogenitorcells)增生、神經遞質(neurotransmitter)、在星狀細胞中刺激發炎細胞激素(cytokines)表現以及在發育過程中吸引神經幹細胞移位(migration)的作用。最近的研究顯示SDF-1在癲癇引發之後的表現量會迅速的上升。我們初步的結果以SDF-1的拮抗劑(antagonist)AMD3100抑制SDF-1作用，會降低引發癲癇後的死亡率，以及減少海馬齒狀回細胞新生數量。這些結果顯示SDF-1在癲癇急性期的發作以及海馬齒狀回細胞新生的過程中扮演重要角色。在此三年計畫中，我們將以電生理學、分子生物學、組織學等方式，利用conditionalSDF-1基因剔除小鼠為動物模式深入探討SDF-1在癲癇後調控海馬迴神經細胞興奮性之關係以及SDF-1調控神經細胞細胞新生之機制如下：目標一：探討SDF-1在癲癇後調控神經興奮性之作用機轉。我們初步的結果顯示抑制SDF-1的作用可以減少引發癲癇後的致死率，我們將利用conditionalSDF-1基因剔除小鼠探討SDF-1在癲癇後在海馬迴中的作用機制和功能。方法：我們將利用SDF-1的agonist以及antagonist刺激正常及SDF-1conditionalknockout小鼠在癲癇後記錄海馬迴中的pyramidalcell突觸間的glutamatetransmissionsystem的作用。經由記錄在AMPAorNMDAmediatedEPSC(EPSCAMPAorEPSCNMDA),paired-pulserecording,miniatureEPSCrecording,exogenousapplicationofglutamate等電生理學方式探討SDF-1對突觸的影響。目標二：探討SDF-1在癲癇後急性期引起細胞激素表現的作用機制與增加神經刺激的功能。SDF-1在離體實驗中可以經由其下游之ERK/Akt蛋白引發細胞激素的表現，細胞激素具有刺激海馬迴神經細胞興奮及引發細胞凋亡的作用。方法：利用SDF-1conditional基因剔除小鼠模型以及組織免疫螢光染色及分子生物學方法探討癲癇之後的免疫相關基因如IL-1b，IL-6及TNF-a等表現與SDF-1作用的因果關係及SDF-1如何透過細胞激素刺激神經興奮及引發細胞凋亡的機制。目標三：探討SDF-1促進癲癇後神經前驅幹細胞增生的機制。SDF-1持續刺激會增加神經前驅細胞的增生。我們目前的結果顯示抑制SDF-1的作用會減少癲癇後海馬齒狀回中神經前驅細胞的增生。在此計畫中我們將探討此一作用的機制。實驗方法：(a)利用在神經幹細胞中特異表現的SOX2-GFP螢光小鼠，以whole-cellpatch-clamp記錄hilar區域中subgranulezone神經前驅幹細胞的神經興奮性是否增加，藉此探討SDF-1在癲癇後是否增加對神經前驅幹細胞的刺激。(b)癲癇後用BrdU標定新生細胞，並且利用SDF-1conditional基因剔除小鼠模型及AMD3100處理方式探討SDF-1在各個時間點促進癲癇後神經前驅細胞增生的作用。(c)以分子生物學方式探討SDF-1在癲癇後對subgranulezone神經前驅幹細胞的ERK及p38訊息傳導路徑的活化作用及其對細胞增生的作用。預期的結果：我們目前的結果顯示以AMD3100阻斷SDF-1的作用可以減少神經前驅細胞增生的數量。這些實驗將有助於了解癲癇之後SDF-1對此一現象的作用機制。目標四：探討SDF-1在癲癇之後吸引顆粒細胞異位移動的作用。SDF-1與CXCR4已知對發育早期的神經幹細胞有導引的作用，SDF-1在癲癇後於海馬迴中的表現量上升。本計畫將探討SDF-1對於顆粒細胞在癲癇之後的吸引作用機制。實驗方法：(a)我們將利用neurosphere的離體模型探討SDF-1的化學引誘物及細胞激素作用。正常的以及刺激過的neurosphere測試其對SDF-1的反應能力。我們也將測試經過SDF-1，glutamate或是pilocarpine刺激的Neurosphere的基因表現。(b)我們將追蹤觀察表現CXCR4的異位移動細胞及其神經細胞標記基因的表現。(c)利用conditionalSDF-1剔除小鼠觀察其顆粒細胞異位移動。SDF-1在正常小鼠或是組織中具有刺激神經、促進正常神經前驅幹細胞增生及在胚胎時期促進granulecell移動的功能。癲癇後小鼠海馬迴中的SDF-1蛋白表現上升，同時發生神經細胞過度興奮、神經前驅幹細胞增生及granulecell異位移動。本計劃將探討SDF-1在此現象中所扮演角色。待本計畫完成後,我們可以了解SDF-1在癲癇病程中所扮演的角色，除了可以增進對癲癇症的了解之外，也可以利用這些結果應用在開發抗癲癇藥物。對於增進癲癇病患福祉將有重大的貢獻。<br> Abstract: Increased neurogenesis and hyper-excitation of neurons are the pathological responses of seizure.However the mechanism remains unclear. SDF-1 is a small molecule secretion protein that canstimulate neuron progenitor cell proliferation, act as a neurotransmitter, promote cytokinesexpression in astrocytes, and attract progenitor cell migration. Recent studies had shown that SDF-1 expression is induced rapidly after seizure in the astrocyte of hippocampus. Blocking the activityof SDF-1 by its antagonist AMD3100 will reduce the mortality after seizure and neurogenesis.These results suggest SDF-1 activity is required for the acute seizure response and progenitor cellproliferation in dentate gyrus. These results suggest that SDF-1 play an important role in the acutephase of epilepsy and the neurogenesis in dentate gyrus. In this project, we will investigate themechanism of SDF-1 in the process of status epilepsy by the methods of electrophysiology,molecular biology and histology with the animal model of SDF-1 conditional knockout. In thisthree-year project, we will investigate the role of SDF-1 in the neuronal excitation andneurogenesis after status epilepsy.Specific Aims:AIM1: To examine the neuronal excitation mechanism of SDF-1 in the acute phase of statusepilepsy. This aim will be performed in the first to third years.Rationale: Our preliminary results show that blocking the activities of SDF-1 will reduce themortality and seizure duration after induction of seizure. We will conditionally knockout theexpression of SDF-1 and assay for the neuronal excitation mechanism of SDF-1 in the acute phaseof status epilepsy.Experimental approaches: We will record the synaptic glutamate transmission of hippocampuswith SDF-1 agonist or antagonist treated normal and SDF-1 conditional knockout mice. The roleof SDF-1 on synaptics will be investigated by the methodology of electrophysiology.AIM2: We will investigate the function and mechanism of SDF-1 in elevation the expressionof cytokines. This aim will be performed in the first to third years.Rationale: In vitro studies show that SDF-1 can promote the expression of cytokines via theErk/Akt signaling pathway. The cytokines are responsible for the neuronal excitation andapoptosis after induction of status epilepsy.Experimental approaches: We will investigate the cytokine expression elevation activity ofSDF-1 in the astrocyte of hippocampus after seizure by the method of molecular biology andimmunohistology with SDF-1 conditional knockout mice.AIM3: To examine the role of SDF-1 in promoting proliferation of neuron progenitor cellafter status epilepsy. This aim will be performed in the first to third years.Rational: Tonic release of SDF-1 protein can promote the proliferation of neuronal progenitorcells. We expect that SDF-1 can promote the excess cell proliferation in the dentate gyrus afterseizure as well. Our preliminary results show that blocking the activity of SDF-1 by its antagonistwill reduce the cell proliferation in the dentate gyrus after induction of status epilepsy. We willinvestigate the mechanism of SDF-1 in promoting the neuron progenitor cell proliferation afterseizure.Experimental approaches:(a) We will use the SOX2-eGFP mice to locate the neuronal progenitor cells and record theelectrophysiological response of neuronal progenitor cells in the subgranule zone by the method ofwhole-cell patch-clamp. This will reveal the SDF-1 excitation role of neuronal progenitor cells.(b) We will label the newly proliferated cells and investigate the progenitor cell proliferationrole of SDF-1.Anticipated results: Our preliminary results show that the neuron progenitor cell proliferation isreduced by administration of AMD3100 after induction of seizure. These experiments will revealthe role of SDF-1 in the increased progenitor cell proliferation after induction of status epilepsy.AIM4: To examine the role of SDF-1 in attracting ectopic granule cell migration afterseizure. This aim will be performed in the first to third years.Rationale:SDF-1/CXCR4 axis is known to be responsible for the neuron stem cell migration in developingstages. SDF-1 expression is elevated after induction of seizure, and the neuron progenitor cellectopic migration is significant consequence of epileptic animals. We would like to investigate therole of SDF-1 in the process of ectopic progenitor cell migration.Experimental approaches:(a) We will test the chemoattractant and cytokine effect of SDF-1 with neurosphere for the invitro model system. We will assay the chemotaxis activity of SDF-1 with normal or excitedneuronal sphere. Neuronal sphere treated with SDF-1, glutamate or pilocarpine will be used foranalysis for the gene expression.(b) We will long-term follow-up the CXCR4 expressing ectopic migrated cells, and neuralmarkers will be traced to verify the identity of the cell type in several time points after inductionof seizure.(c) Test the ectopic granule cell migration with conditional targeted SDF-1.(d) Conditional knockout of SDF-1 will provide an excellent tool for an SDF-1 nullenvironment. Application with proper Cre expressing mice strain, we will be able to investigatethe regional and temporal requirement of SDF-1 for ectopic progenitor cell migration.Anticipated results:Our preliminary results show that SDF-1 protein is capable to induce migration of cultured neuronprogenitor cells. These experiments will reveal that if excitation of neuronal sphere will alter itsresponse to SDF-1.SDF-1 is capable to promoteneuronal excitation, neuronalprogenitor cell proliferation, andgranule cell migration in normalmice. The expression of SDF-1 iselevated in epileptic brains of theregions coincide with the overexcitationexcess neuronalprogenitor proliferation and granulecells ectopic hilar migration. Wewill investigate the above roles ofSDF-1 after epilepsy.Conclusion: This project will investigate the role of SDF-1 in the epileptogenesis. The results willnot only increase the knowledge of status epilepsy but also developing new drugs for statusepilepsy patients.癲癇海馬迴神經細胞新生epilepsyneurogenesishippocampus