本文選題：驚厥持續(xù)狀態(tài)　切入點(diǎn)：海馬　出處：《重慶醫(yī)科大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分持續(xù)驚厥后星形膠質(zhì)細(xì)胞活化與神經(jīng)元損傷的動態(tài)觀察 目的：觀察驚厥持續(xù)狀態(tài)后大鼠海馬星形膠質(zhì)細(xì)胞與神經(jīng)元的動態(tài)變化。 方法：以成年SD大鼠為研究對象，建立氯化鋰-匹羅卡品誘導(dǎo)60minSC模型，在SC后1天至8周的4個時間點(diǎn)上采集大鼠海馬組織，采用免疫熒光雙重標(biāo)記GFAP/NeuN，觀察SC后大鼠海馬星形膠質(zhì)細(xì)胞與神經(jīng)元的動態(tài)變化；采用RT-qPCR與Western Blot檢測GFAP的表達(dá)。 結(jié)果：（1）以NeuN標(biāo)記神經(jīng)元發(fā)現(xiàn)，SC后1天大鼠海馬出現(xiàn)以CA1區(qū)、CA3區(qū)為著的神經(jīng)元死亡，持續(xù)至SC后8周，并可見神經(jīng)元丟失遺留空穴；（2）GFAP標(biāo)記的星形膠質(zhì)細(xì)胞于SC后5天出現(xiàn)數(shù)目增多（P0.05），持續(xù)至SC后8周（P0.01），并可見細(xì)胞胞體肥大，，突起增多增粗，在神經(jīng)元丟失區(qū)域形成膠質(zhì)疤痕；（3）GFAP-mRNA的表達(dá)水平在SC后1天即有升高，在SC后5天、SC后4周、SC后8周均顯著高于正常對照組（P0.01），于SC后4周達(dá)高峰；（4）SC后5天GFAP的蛋白表達(dá)水平開始升高，SC后4周達(dá)高峰，SC后8周仍高于正常對照組（P0.01），與GFAP-mRNA的表達(dá)基本一致。 結(jié)論：SC后大鼠海馬出現(xiàn)選擇性神經(jīng)元壞死及反應(yīng)性星形膠質(zhì)細(xì)胞活化，一直持續(xù)至慢性癲癇晚期；膠質(zhì)活化開始于SC后潛伏期，早于慢性癲癇期。 第二部分：星形膠質(zhì)細(xì)胞介導(dǎo)腺苷信號通路在持續(xù)驚厥后的表達(dá)變化 目的：探討星形膠質(zhì)細(xì)胞介導(dǎo)腺苷信號通路在癲癇形成中的動態(tài)變化。 方法：以成年SD大鼠為研究對象，建立氯化鋰-匹羅卡品誘導(dǎo)60minSC模型，在SC后1天至8周的4個時間點(diǎn)上采集大鼠海馬組織，采用高效液相色譜法檢測海馬腺苷含量的動態(tài)變化；采用免疫熒光雙重標(biāo)記ADK/GFAP、ADK/NeuN，檢測SC后大鼠海馬星形膠質(zhì)細(xì)胞與神經(jīng)元中ADK的表達(dá)；采用RT-qPCR與Western Blot檢測ADK的表達(dá)；采用Western Blot檢測腺苷受體A1R、A2aR、A2bR、A3R的表達(dá)。 結(jié)果：（1）與正常對照組相比，SC后1天海馬腺苷含量升高并達(dá)高峰（P0.01），在SC后5天開始下降，SC后4周、SC后8周明顯回落，SC后8周顯著低于正常對照組（P0.01）；（2）SC后5天ADK/GFAP陽性細(xì)胞數(shù)開始增多（P0.01），于SC后4周達(dá)高峰，并持續(xù)至SC后8周， ADK在星形膠質(zhì)細(xì)胞胞漿中表達(dá)明顯；SC后5天ADK/NeuN陽性細(xì)胞數(shù)目開始增多，于SC后8周達(dá)高峰（P0.01），主要分布于海馬CA1區(qū)、CA3區(qū)，ADK在神經(jīng)元胞核內(nèi)表達(dá)明顯；（3）海馬ADK-mRNA與ADK蛋白表達(dá)水平基本一致，在SC后5天開始升高（P0.01），于SC后4周達(dá)高峰，一直持續(xù)至SC后8周；（4）腺苷受體的表達(dá)呈多樣性，A1R呈雙向表達(dá)，SC后1天較正常對照組明顯升高（P0.01），SC后5天開始下降，并延續(xù)到SC后4周、SC后8周；A2aR的表達(dá)在SC后1天開始升高，于SC后4周達(dá)高峰，一直持續(xù)至SC后8周（P0.01）；A2bR、A3R表達(dá)在SC后1天均開始下降，低于正常對照組（P0.01），一直持續(xù)至SC后8周。 結(jié)論：SC后大鼠海馬腺苷含量在急性期急劇升高，但未能維持至慢性癲癇期，可能與ADK的表達(dá)逐漸增強(qiáng)有關(guān)；腺苷及腺苷受體A1R/A2aR表達(dá)失衡，可能削弱腺苷的內(nèi)源性抗驚厥作用，促進(jìn)癲癇發(fā)生。 第三部分:星形膠質(zhì)細(xì)胞介導(dǎo)的腺苷信號與驚厥易感性的實驗研究 目的：探討星形膠質(zhì)細(xì)胞介導(dǎo)的腺苷信號通路在癲癇形成中的作用及可能機(jī)制。 方法：采用膜片鉗記錄技術(shù)，以無鎂誘導(dǎo)海馬腦片放電為模型，的影響；采用星形膠質(zhì)細(xì)胞—神經(jīng)元共培養(yǎng)體系，免疫熒光雙重標(biāo)記ADK/GFAP、ADK/NeuN，觀察ADK在原代培養(yǎng)神經(jīng)細(xì)胞中的分布和表達(dá)特點(diǎn)；采用膜片鉗記錄技術(shù)，以無鎂誘導(dǎo)神經(jīng)元放電為模型，檢測ADK抑制劑、A1R拮抗劑、A2aR拮抗劑對神經(jīng)元興奮性的影響。 結(jié)果：（1）與正常人工腦脊液海馬腦片相比，無鎂人工腦脊液可誘導(dǎo)海馬腦片爆發(fā)樣的自發(fā)性放電，ADK抑制劑、腺苷A2aR拮抗劑可引起無鎂誘導(dǎo)海馬腦片的爆發(fā)性放電的潛伏期延長，放電持續(xù)時間縮短（P0.01），A1R拮抗劑未能改變無鎂人工腦脊液海馬腦片的放電特性（P0.05）；（2）體外分離培養(yǎng)的海馬神經(jīng)元與星形膠質(zhì)細(xì)胞均有ADK表達(dá)，ADK在神經(jīng)元中呈核型分布，在星形膠質(zhì)細(xì)胞中呈胞漿型與核型共同分布；（3）與正常細(xì)胞外液神經(jīng)元的動作電位相比，無鎂細(xì)胞外液可誘導(dǎo)神經(jīng)元異常放電，作用于星形膠質(zhì)細(xì)胞的ADK抑制劑使神經(jīng)元異常放電頻率明顯下降（P0.01），而單純作用于神經(jīng)元的ADK抑制劑對神經(jīng)元異常放電頻率無明顯改變（P0.05）；（4）A2aR拮抗劑可降低神經(jīng)元異常放電頻率(P0.01），而A1R拮抗劑未能減少神經(jīng)元異常放電頻率（P0.05）。 結(jié)論：ADK通過星形膠質(zhì)細(xì)胞調(diào)節(jié)腺苷代謝，ADK抑制劑可降低神經(jīng)元的興奮性，提高驚厥的閾值；腺苷抗驚厥特性可能主要由A1R介導(dǎo)，而在A2aR水平的調(diào)控也有助于調(diào)節(jié)神經(jīng)興奮性。
[Abstract]:The dynamic observation of astrocyte activation and neuron damage in the first part after persistent convulsion
Objective: To observe the dynamic changes of astrocytes and neurons in the hippocampus of rats after the persistent state of convulsion.
Methods: adult SD rats as the research object, the establishment of lithium pilocarpine induced 60minSC model rat hippocampus, collected at 4 time points SC after 1 and 8 weeks, using double immunofluorescent labeling of GFAP/NeuN, the dynamic changes of rat hippocampal astrocytes and neurons were observed by SC; expression RT-qPCR Western and Blot GFAP detection.
Results: (1) using NeuN labeled neurons, 1 days after SC in the hippocampus of rats in CA1 District, CA3 district for the death of neurons, until 8 weeks after SC, neuronal loss and visible left hole; (2) GFAP labeled astrocytes in SC occurred 5 days after the increase of the number of (P0.05), until 8 weeks after SC (P0.01), and visible cell body hypertrophy, prominences thickening in regional neuronal loss formation of glial scar; (3) the expression level of GFAP-mRNA in SC after 1 days increased in SC after 5 days, 4 weeks after SC, SC after 8 weeks significantly higher than the normal control group (P0.01), 4 weeks after SC and reached the peak 5 days; (4) the protein expression level of GFAP began to increase after SC, the peak of up to 4 weeks after SC, SC after 8 weeks is still higher than the normal control group (P0.01), consistent with the expression of GFAP-mRNA.
Conclusion: after SC, the selective neuron necrosis and reactive astrocyte activation in the hippocampus continue to reach the late stage of chronic epilepsy in rats. The activation of glial cells starts after SC, which is earlier than the chronic epilepsy stage.
The second part: the changes in the expression of adenosine signaling pathway mediated by astrocytes after persistent convulsion
Objective: To investigate the dynamic changes of astrocyte mediated adenosine signal pathway in the formation of epilepsy.
Methods: adult SD rats as the research object, the establishment of lithium pilocarpine induced 60minSC model rat hippocampus, collected at 4 time points SC after 1 and 8 weeks, the dynamic changes of the HPLC method for the determination of adenosine in hippocampus; using double immunofluorescence labeling of ADK/GFAP and ADK/NeuN. The expression of SC after rat hippocampal astrocytes and neurons in ADK; the expression of RT-qPCR and Western Blot detection of ADK by Western Blot; detection of adenosine receptor A1R, A2aR, A2bR, A3R expression.
緇撴灉錛

本文編號：1589659