【作者】 解媛媛；

【导师】 肖波；

【作者基本信息】 中南大学 ， 神经病学， 2012， 博士

【摘要】 目的观察大鼠癫痫持续状态(status epilepticus, SE)后海马区脑组织miR-34a的表达分布规律；通过干预miR-34a表达水平,探讨其在癫痫后海马神经元凋亡中的作用。方法1.建立模型：采用6-8周龄健康雄性SD大鼠,随机分为空白对照组(Normal, N组),癫痫组(Epilepsy, Ep组),干预组(Antagomir,A组)和干预对照组(Antagomir-control,C组)。对EP组、A组、C组建立氯化锂-匹罗卡品致痫大鼠SE模型。在SE诱导成功后4-6小时分别对A组和C组侧脑室立体定向给予Antagomir和Antagomir-control干预miR-34a表达水平,各组均以在1天(24小时,急性期)、7天(静止期)作为研究时间点。2.对大鼠行脑电图检查,记录生理状态、致痫期Ⅳ级以上发作状态、造模成功后发作间期状态、自发发作状态的脑电变化,确认癫痫模型的建立。3.实时定量-PCR检测各组miR-34a表达水平,检测干预前后miR-34a表达量的变化,以验证药物干预miR-34a的效果。4.采用Western Blot和免疫组化技术检测caspase-3蛋白在大鼠海马组织中的分部及其表达量的变化。5. TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling)法检测大鼠SE后海马区脑组织神经元细胞凋亡情况。6. Nissl染色研究大鼠SE后海马区脑组织神经元细胞缺失及存留情况。结果1.依据动物行为学表现及脑电图结果,最终造模组(Ep组、A组、C组)大鼠成模率为89.17%。成模及干预后,大鼠因自身恢复、麻醉意外、手术创伤等因素,部分发生死亡,最终可用实验组动物的成活率为69.17%。2.实时定量PCR检测显示,在急性期和静止期,miR-34a的表达在Ep组较N组均显著增高,药物干预后A组较C组均显著降低,有显著性差异,干预有效。3. Western Blot检测显示,在急性期和静止期,caspase-3蛋白的表达在Ep组较N组均显著增高,药物干预后A组较C组均显著降低,有显著性差异,与实时定量PCR结果趋势相一致。4.免疫组化检测显示,在急性期和静止期,Ep组与药物干预对照C组中caspase-3蛋白在海马(CA1、CA3区)神经元胞浆均高表达,而N组和药物干预A组均仅有较低密度的表达,与Western Blot结果相一致。5. TUNEL检测显示,在急性期和静止期,Ep组海马(CA1、CA3区)神经元细胞凋亡均明显多于N组；药物干预后A组海马(CA1、CA3区)神经元细胞凋亡均明显低于C组,与实时定量PCR、Western Blot、免疫组化结果相一致。6. Nissl染色显示,在急性期和静止期,Ep组海马(CA1、CA3区)神经元细胞损失均明显多于N组,存留的神经元细胞均明显少于N组；药物干预后A组海马(CA1、CA3区)神经元细胞损失均明显少于C组,存留的神经元细胞均明显多于N组,与实时定量PCR、Western Blot、免疫组化、TUNEL结果相适应。结论大鼠癫痫持续状态后的急性期和静止期,海马(CA1、CA3区)神经元中miRNA-34a可通过上调caspase-3蛋白的表达,激活下游凋亡通路,造成海马区神经元细胞凋亡。更多还原

【Abstract】 ObjectiveTo study the expressional change of miR-34a in rat hippocampus following status epilepticus (SE). Through intervention of expressional level of miR-34a, this research was also aimed to explore the role of miR-34a and its mechanism in neuronal apoptosis in rats following SE.Method1. SE Model:6-8week, healthy male SD rats were randomly divided into the negative control group (Normal, N group), the epilepsy group (epilepsy, Ep group), the intervention group (Antagomir, A group) and the intervention control group (Antagomir-control, C group). Then the lithium chloride-pilocarpine SE model was established, A and C groups.4-6hours after models set, Antagomir and Antagomir-control were injected into rats’lateral ventricle to intervene miR-34a level in A and C groups by animal brain stereotactic skills. Then corresponding indicators in1day (24hours, Acute phase) and7days (Quiescent) after SE were observed.2. The experimental rats EEG for physiological state, SE, interictal state and spontaneous onset state were recorded to verify that the models were set successfully. 3. Quantitative real-time PCR(qRT-PCR) detection was performed to study the miR-34a expression level in rats after SE.4. Western blot and Immunohistochemical detection were used to detect the expression level of caspase-3protein.5. TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling) was used to detect hippocampal neuronal apoptosis in experimental rats.6. Nissl staining was exploited to detect hippocampal neuronal loss and retention in experimental rats.Result1. Based on animal behavior and EEG results, a successful rate of89.7%for SE rats modules was made in A and C groups. After modules set and intervention, there were69.17%rats (Ep, A and C groups) alive. Meanwhile the cause of death mainly included restored ability, anesthesia, surgical trauma and other factors.2. qRT-PCR results showed that the expression change of miR-34a at acute phase and quiescent were in consistent trend. Ep group’s miR-34a level was significantly higher than N group’s, whereas A group’s miR-34a level was significantly lower than C group’s.3. Western blot results showed that expression of caspase-3protein at acute phase and quiescent were in consistent trend. The caspase-3 protein level in Ep group was significantly higher than N group’s, whereas A group’s caspase-3protein level was significantly lower than C group’s, which was consistent with the results of qRT-PCR.4. Immunohistochemical results showed that expression of caspase-3protein at acute phase and quiescent were in consistent trend. The expression level of caspase-3protein in Ep group was significantly higher than N group’s, whereas A group’s caspase-3protein level was significantly lower than C group, which was consistent with qRT-PCR and Western blot results.5. TUNEL results showed the apoptotic neurons at acute phase and quiescent were in consistent trend. The hippocampal neuron apoptosis of Ep group was significantly higher than that of N group, whereas the hippocampal apoptotic neurons of A group was significantly lower than its counterpart in C group.6. Nissl staining results showed the survival and loss of neurons at acute phase and quiescent were in consistent trend. The loss of hippocampal neurons in Ep group was significantly higher than in N group, wheras survival of hippocampus neurons in Ep group was significantly lower than that in N group. The loss of rat hippocampal neurons in A group was significantly lower than that in C group, while survival of hippocampal neurons in A group was significantly higher than that in C group. ConclusionAfter mice SE (at acute phase and quiescent), miR-34a in hippocampal neuron could increase the expression of caspase-3, activate the downstream pathway of apoptosis, and lead to hippocampal neuron loss.更多还原