【摘要】：第一部分體外培養(yǎng)海馬神經(jīng)元網(wǎng)絡(luò)癇性電活動(dòng)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(luò)中,應(yīng)用4-AP(50μM)分別進(jìn)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(jìn)行比較,建立4-AP的致癇神經(jīng)網(wǎng)絡(luò)模型。觀察兩種模型上神經(jīng)元癇樣放電的情況,為進(jìn)一步探討癲癇的能量代謝特征提供神經(jīng)網(wǎng)絡(luò)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(xì)胞染色鑒定神經(jīng)元。培養(yǎng)至第8天采用全細(xì)胞膜片鉗技術(shù)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(luò)。培養(yǎng)第8～10d時(shí)細(xì)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(jìn)行膜片鉗檢測,發(fā)現(xiàn)兩組培養(yǎng)細(xì)胞均出現(xiàn)高頻與高幅度癇樣放電。在5-10min時(shí)間段達(dá)到高峰后逐漸下降,在每個(gè)時(shí)間段的放電頻率及平均振幅均遠(yuǎn)遠(yuǎn)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(jì)學(xué)意義。三組間的平均振幅及放電頻率在各時(shí)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(luò)模型,作為體外研究癇性網(wǎng)絡(luò)的工具。 第二部分體外培養(yǎng)胚胎海馬神經(jīng)元網(wǎng)絡(luò)的能量代謝特征及與癇性標(biāo)志變化間的關(guān)系 目的在體外培養(yǎng)的大鼠胚胎海馬神經(jīng)元細(xì)胞上,建立低鎂及4-AP誘發(fā)癇性神經(jīng)網(wǎng)絡(luò)模型,檢測30min內(nèi)線粒體膜電位的變化,及細(xì)胞能量代謝標(biāo)志分子ATP, ADP濃度,及網(wǎng)絡(luò)致癇標(biāo)志分子細(xì)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(luò)線粒體膜電位水平、與能量代謝及網(wǎng)絡(luò)致癇狀態(tài)的關(guān)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(luò)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(luò)中Glu及GABA濃度。 結(jié)果大鼠胚胎海馬神經(jīng)元網(wǎng)絡(luò)誘導(dǎo)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(luò)誘導(dǎo)SLEs的事件可視為神經(jīng)元網(wǎng)絡(luò)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(biāo)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(luò)誘導(dǎo)SLEs后呈現(xiàn)規(guī)律性時(shí)相變化。在此過程中,細(xì)胞線粒體膜電位與癇性活動(dòng)標(biāo)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(luò)誘導(dǎo)SLEs的事件可視為神經(jīng)元網(wǎng)絡(luò)高電位爆發(fā)過程,并伴有高代謝特征。但相對而言,癇性標(biāo)志分子的變化較電位變化遲滯。圖21幅,表16個(gè),參考文獻(xiàn)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(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R742.1;R-332

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