【摘要】：第一部分體外培養(yǎng)海馬神經(jīng)元網(wǎng)絡癇性電活動模型的建立 目的在體外培養(yǎng)原代大鼠胚胎海馬神經(jīng)元網(wǎng)絡中,應用4-AP(50μM)分別進行致癇,通過與經(jīng)典的低鎂海馬神經(jīng)元癇樣放電(SLEs)模型進行比較,建立4-AP的致癇神經(jīng)網(wǎng)絡模型。觀察兩種模型上神經(jīng)元癇樣放電的情況,為進一步探討癲癇的能量代謝特征提供神經(jīng)網(wǎng)絡模型。 方法取孕第18-19天的胚胎SD大鼠,分離海馬神經(jīng)元培養(yǎng),在倒置相差顯微鏡下觀察神經(jīng)元的生長情況。采用Map2免疫熒光細胞染色鑒定神經(jīng)元。培養(yǎng)至第8天采用全細胞膜片鉗技術記錄神經(jīng)元在無鎂培養(yǎng)液、4-AP處理不同后30main內(nèi)的放電情況。 結(jié)果神經(jīng)元在種植24h后大部分貼壁,突起逐漸增多,在神經(jīng)元間逐漸互相連接形成網(wǎng)絡。培養(yǎng)第8～10d時細胞最為豐滿。第8天的神經(jīng)元經(jīng)無鎂培養(yǎng)液或4-AP處理后馬上進行膜片鉗檢測,發(fā)現(xiàn)兩組培養(yǎng)細胞均出現(xiàn)高頻與高幅度癇樣放電。在5-10min時間段達到高峰后逐漸下降,在每個時間段的放電頻率及平均振幅均遠遠高于正常對照組(p0.001)。而低鎂及4-AP兩種致癇模型的比較,平均振幅及放電頻率在30min內(nèi)差異無統(tǒng)計學意義。三組間的平均振幅及放電頻率在各時間變化均有顯著性差異。 結(jié)論體外培養(yǎng)的胚胎海馬神經(jīng)元經(jīng)4-AP處理后,可誘發(fā)出與低鎂處理一樣的高頻與高幅度癇樣放電,可視為一種體外癇性神經(jīng)網(wǎng)絡模型,作為體外研究癇性網(wǎng)絡的工具。 第二部分體外培養(yǎng)胚胎海馬神經(jīng)元網(wǎng)絡的能量代謝特征及與癇性標志變化間的關系 目的在體外培養(yǎng)的大鼠胚胎海馬神經(jīng)元細胞上,建立低鎂及4-AP誘發(fā)癇性神經(jīng)網(wǎng)絡模型,檢測30min內(nèi)線粒體膜電位的變化,及細胞能量代謝標志分子ATP, ADP濃度,及網(wǎng)絡致癇標志分子細胞外谷氨酸(Glu)及γ氨基丁酸(GABA)的含量。觀察致癇神經(jīng)網(wǎng)絡線粒體膜電位水平、與能量代謝及網(wǎng)絡致癇狀態(tài)的關系。 方法分別用低鎂培養(yǎng)液及4-AP對培養(yǎng)的海馬神經(jīng)元處理Omin,5min,1Omin,15min,20min,25min,30min后,利用熒光分光光度法檢測胚胎海馬神經(jīng)元網(wǎng)絡線粒體膜電位,用比色法檢測海馬神經(jīng)元內(nèi)ATP、 ADP含量,用ELISA法檢測海馬神經(jīng)網(wǎng)絡中Glu及GABA濃度。 結(jié)果大鼠胚胎海馬神經(jīng)元網(wǎng)絡誘導SLEs后呈,線粒體膜電位、ADP及谷氨酸短期內(nèi)升高,之后逐漸降低；而ATP及GABA則呈現(xiàn)相反變化。表明神經(jīng)元網(wǎng)絡誘導SLEs的事件可視為神經(jīng)元網(wǎng)絡的高電位爆發(fā)過程,并伴有高代謝特征。但相對而言,癇性標志分子的變化較電位變化遲滯。 結(jié)論大鼠胚胎海馬神經(jīng)元網(wǎng)絡誘導SLEs后呈現(xiàn)規(guī)律性時相變化。在此過程中,細胞線粒體膜電位與癇性活動標志分子Glu呈現(xiàn)先升后降,而ATP與GABA呈現(xiàn)先降后升的變化趨勢。這表明神經(jīng)元網(wǎng)絡誘導SLEs的事件可視為神經(jīng)元網(wǎng)絡高電位爆發(fā)過程,并伴有高代謝特征。但相對而言,癇性標志分子的變化較電位變化遲滯。圖21幅,表16個,參考文獻42篇
[Abstract]:The first part is the establishment of epileptic electrical activity model of hippocampal neuron network cultured in vitro. Objective to induce eclampsia by 4-AP (50 渭 M) in primary rat embryonic hippocampal neuron network in vitro. Compared with the classical epileptic discharge (SLEs) model of low magnesium hippocampal neurons, the epileptic neural network model of 4-AP was established. To observe the epileptic discharge of neurons in the two models, and to provide a neural network model for further study of the characteristics of energy metabolism in seizures. Methods the hippocampal neurons were isolated from embryonic SD rats on the 18th and 19th day of gestation, and the growth of neurons was observed under inverted phase contrast microscope. The neurons were identified by Map2 immunofluorescence cell staining. On the 8th day, the discharge of neurons in magnesium-free medium and 30main treated with 4-AP was recorded by whole-cell patch clamp technique. Results after 24 hours of implantation, most of the neurons adhered to the wall, the processes increased gradually, and the neurons gradually connected to each other to form a network. The cells were the most plump on the 8th to 10th day of culture. On the 8th day, the neurons were treated with magnesium-free medium or 4-AP as soon as they were treated with patch clamp. It was found that the cells in both groups showed high frequency and high amplitude epileptic discharge. After reaching the peak in 5-10min time period, the discharge frequency and average amplitude were much higher than those in the normal control group (p0. 001), and the discharge frequency and average amplitude in each time group were much higher than those in the normal control group (p0. 001). However, there was no significant difference in average amplitude and discharge frequency between low magnesium and 4-AP epileptic models in 30min. There were significant differences in average amplitude and discharge frequency among the three groups at each time. Conclusion the embryonic hippocampal neurons cultured in vitro can induce the same high frequency and high amplitude epileptic discharge as low magnesium treatment after 4-AP treatment, which can be regarded as a epileptic neural network model in vitro and used as a tool to study epileptic network in vitro. The second part is the energy metabolism characteristics of embryonic hippocampal neuron network cultured in vitro and its relationship with the changes of epileptic markers. Objective to investigate the relationship between energy metabolism and epileptic markers in rat embryonic hippocampal neuron cells cultured in vitro. The neural network model of epileptic induced by low magnesium and 4-AP was established. The changes of mitochondrial membrane potential and the concentration of ATP, ADP, a marker of cell energy metabolism, were detected in 30min. And the contents of extracellular glutamic acid (Glu) and gamma aminobutyric acid (GABA) in eclampsia induced by the network. To observe the relationship between the level of mitochondrial membrane potential and energy metabolism and the state of eclampsia induced by epileptic neural network. Methods the cultured hippocampal neurons were treated with Omin,5min,1Omin,15min,20min,25min,30min in low magnesium medium and 4-AP, respectively. the mitochondrial membrane potential of embryonic hippocampal neuron network was measured by fluorescence spectrophotometry. The content of ATP, ADP in hippocampal neurons was measured by colorimetric method, and the concentrations of Glu and GABA in hippocampal neural network were detected by ELISA method. Results after SLEs induced by rat embryonic hippocampal neuron network, the mitochondrial membrane potential, ADP and glutamic acid increased in a short period of time, and then decreased gradually, while ATP and GABA showed opposite changes. It is suggested that the event of SLEs induced by neural network can be regarded as the process of high potential burst of neural network, accompanied by high metabolic characteristics. But relatively speaking, the change of epileptic marker molecules is slower than the change of potential. Conclusion SLEs induced by embryonic hippocampal neuron network in rats shows regular temporal changes. In this process, the mitochondrial membrane potential and epileptic activity marker Glu increased at first and then decreased, while ATP and GABA decreased at first and then increased. This suggests that the event of SLEs induced by neural network can be regarded as a process of high potential burst of neural network, accompanied by high metabolic characteristics. But relatively speaking, the change of epileptic marker molecules is slower than the change of potential. Figure 21, table 16, references 42
【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;R-332

【相似文獻】

相關期刊論文 前10條

1 蔡申甌;陶樂天;;神經(jīng)元網(wǎng)絡動力學的統(tǒng)計物理方法(英文)[J];生理學報;2011年05期

2 陳武凡;神經(jīng)元網(wǎng)絡的信息處理方式與應用[J];第一軍醫(yī)大學學報;1992年01期

3 顧凡及，張濵，，章惠明;一種修正的動態(tài)神經(jīng)元網(wǎng)絡模型的類腦電活動及其應用[J];生物物理學報;1995年04期

4 余生晨;高立;薛陽;黃江蘭;余桂賢;崔新偉;;基于遺傳算法和神經(jīng)元網(wǎng)絡的心電信號T波檢測[J];中國生物醫(yī)學工程學報;2008年04期

5 陳琳;潘海鴻;黃江;;基于互相關函數(shù)的培養(yǎng)神經(jīng)元網(wǎng)絡動態(tài)特征分析[J];生物醫(yī)學工程研究;2008年03期

6 張軍;朱虹;許朝暉;梁剛;季瑞瑞;;神經(jīng)元網(wǎng)絡容錯學習在腎小球區(qū)域邊界增強中的應用[J];生物醫(yī)學工程學雜志;2006年03期

7 陳文娟;李向?qū)?馮顯;蒲江波;駱清銘;;體外培養(yǎng)神經(jīng)元網(wǎng)絡功能結(jié)構的長時間發(fā)育變化[J];科學通報;2010年25期

8 朱耿;蒲江波;孫晶;李向?qū)?;培養(yǎng)海馬神經(jīng)元網(wǎng)絡對刺激位置的群體編碼[J];現(xiàn)代生物醫(yī)學進展;2009年22期

9 楊彩琴;吳國棟;郝敦元;;一維Theta-神經(jīng)元網(wǎng)絡中規(guī)則單放電行波解的進一步討論[J];內(nèi)蒙古大學學報(自然科學版);2010年02期

10 李安安;龔輝;;光學顯微水平全腦成像方法的研究進展[J];生物化學與生物物理進展;2012年06期

相關會議論文 前10條

1 王青云;;時滯對神經(jīng)元網(wǎng)絡時空同步的作用[A];第三屆全國動力學與控制青年學者研討會論文摘要集[C];2009年

2 王青云;;神經(jīng)元網(wǎng)絡系統(tǒng)的非線性時空動力學[A];第四屆全國動力學與控制青年學者研討會論文摘要集[C];2010年

3 孫曉娟;;噪聲激勵下多層次神經(jīng)元網(wǎng)絡放電的動力學行為特性研究[A];第七屆全國動力學與控制青年學者研討會論文摘要集[C];2013年

4 鄭艷紅;陸啟韶;;噪聲影響下的格子耦合神經(jīng)元網(wǎng)絡的斑圖和同步[A];第八屆全國動力學與控制學術會議論文集[C];2008年

5 張蕊;向永光;;神經(jīng)元網(wǎng)絡在唐鋼熱軋1700生產(chǎn)線參數(shù)優(yōu)化中的應用[A];全國冶金自動化信息網(wǎng)2010年年會論文集[C];2010年

6 王青云;;時滯對神經(jīng)元網(wǎng)絡時空同步的作用[A];中國力學學會學術大會'2009論文摘要集[C];2009年

7 李玉葉;張慧敏;魏春玲;楊明浩;王青云;古華光;任維;;隨機信號在神經(jīng)元網(wǎng)絡中誘發(fā)的多次空間相干共振[A];第十二屆全國非線性振動暨第九屆全國非線性動力學和運動穩(wěn)定性學術會議論文集[C];2009年

8 沈瑜;侯中懷;;從簇放電振幅推測耦合神經(jīng)元網(wǎng)絡的度分布性質(zhì)[A];第五屆全國復雜網(wǎng)絡學術會議論文（摘要）匯集[C];2009年

9 李麗明;王眾嫭;王延章;;一種類神經(jīng)元網(wǎng)絡模型及其學習算法初探[A];全國青年管理科學與系統(tǒng)科學論文集（第1卷）[C];1991年

10 陳劍飛;王一玲;林偉;;用神經(jīng)元網(wǎng)絡模型求解多目標決策問題[A];全國青年管理科學與系統(tǒng)科學論文集（第2卷）[C];1993年

相關重要報紙文章 前4條

1 ;GF Srl開發(fā)出用于包裝過程檢測的系統(tǒng)[N];中國包裝報;2005年

2 老鬼阿定;“深度學習”技術進展令人振奮[N];計算機世界;2014年

3 王瀟瀟 楊義勇;我國研發(fā)高分辨“腦地圖”可視儀[N];光明日報;2013年

4 編譯 王喜文;人工智能與大數(shù)據(jù)怎樣結(jié)合？[N];中國電子報;2014年

相關博士學位論文 前10條

1 李玉葉;三類神經(jīng)元網(wǎng)絡的時空動力學行為研究[D];陜西師范大學;2012年

2 賈晨輝;基于辨識與控制的神經(jīng)元網(wǎng)絡模型特性研究[D];天津大學;2012年

3 李向?qū)?基于多微電極陣列的培養(yǎng)神經(jīng)元網(wǎng)絡特性初探[D];華中科技大學;2007年

4 李艷玲;培養(yǎng)神經(jīng)元網(wǎng)絡的學習模型構建及機理研究[D];華中科技大學;2007年

5 盧梅麗;基于相響應的神經(jīng)元網(wǎng)絡同步的優(yōu)化控制[D];天津大學;2013年

6 于海濤;神經(jīng)元網(wǎng)絡的同步、共振及控制研究[D];天津大學;2012年

7 陳傳平;基于多電極陣列的培養(yǎng)神經(jīng)元網(wǎng)絡動態(tài)特征分析[D];華中科技大學;2007年

8 郭大慶;復雜神經(jīng)系統(tǒng)中信息傳輸及信息處理的若干問題研究[D];電子科技大學;2011年

9 陳琳;培養(yǎng)神經(jīng)元網(wǎng)絡自發(fā)電信號的特性分析[D];華中科技大學;2008年

10 曾賢強;基于神經(jīng)元網(wǎng)絡的SFC注入束運線自動調(diào)束控制系統(tǒng)研究[D];蘭州大學;2012年

相關碩士學位論文 前10條

1 時t@;數(shù)字神經(jīng)元網(wǎng)絡的實現(xiàn)與分析研究[D];天津大學;2009年

2 鄭群現(xiàn);具有不可靠突觸的神經(jīng)元網(wǎng)絡的同步研究[D];電子科技大學;2011年

3 王夢松;RBF神經(jīng)元網(wǎng)絡的研究及其在復雜化學信息處理中的應用[D];浙江大學;2002年

4 寧維蓮;耦合神經(jīng)元網(wǎng)絡的隨機動力學行為研究[D];廣西師范大學;2012年

5 賈晨輝;神經(jīng)元網(wǎng)絡同步的分析與控制[D];天津大學;2010年

6 蔡文秀;模糊神經(jīng)元網(wǎng)絡在過程控制中的應用[D];北京化工大學;2006年

7 徐波;時間周期耦合時滯神經(jīng)元網(wǎng)絡動力學行為的研究[D];魯東大學;2014年

8 謝桐瑜;基于過程神經(jīng)元網(wǎng)絡的時序預測模型研究[D];大慶石油學院;2008年

9 肖倩;泛函神經(jīng)元網(wǎng)絡遞歸和剪枝學習算法及其集成研究[D];廣西大學;2013年

10 張維安;培養(yǎng)神經(jīng)元網(wǎng)絡信號處理軟件設計與實現(xiàn)[D];華中科技大學;2007年

本文編號：2486450