【作者】 周成；

【导师】 王净净；

【作者基本信息】 湖南中医药大学 ， 中西医结合临床， 2010， 硕士

【摘要】 [目的]观察愈痫灵颗粒对红藻氨酸(KA)致颞叶癫痫大鼠学习记忆能力及海马组织MFS(苔藓纤维出芽)的影响并研究其相关机制。[方法]通过立体定位仪向大鼠海马局部注射KA而建立颞叶癫痫模型,诱发大鼠癫痫发作,以大鼠水迷宫试验的学习、记忆成绩来评价愈痫灵颗粒对致痫鼠学习记忆能力的影响；采用透射电镜观察海马CA3区超微结构变化；运用Timm硝酸银染色法观察海马苔藓纤维出芽情况；以免疫组化法检测苔藓纤维出芽标志物生长相关蛋白(GAP43)的变化,来探讨愈痫灵颗粒对KA致颞叶癫痫大鼠学习记忆能力与海马组织MFS(苔藓纤维出芽)的影响并研究其相关机制。[结果]1.水迷宫实验结果愈痫灵组、拉莫三嗪组学习记忆成绩明显提高,与空白对照组比较,学习、记忆成绩差异无统计学意义(P>0.05),与模型组比较,学习、记忆成绩差异均具有统计学意义(P<0.05)；愈痫灵组与拉莫三嗪组比较差异无统计学意义(P>0.05),提示愈痫灵颗粒、拉莫三嗪可明显改善癫痫模型大鼠学习记忆能力。2.透射电镜观察海马CA3区超微结构结果空白对照组海马组织CA3区神经元细胞核呈圆形,核膜结构清晰,胞质内可见线粒体、粗面内质网、滑面内质网和大量的多核糖体。假手术组胶质细胞附近有局灶水肿。模型组核膜固缩、轴缩样变性,胞质内细胞器不清,异染色质增多、边聚明显,神经元细胞附近大范围水肿。胶质细胞局部水肿明显。血管内皮细胞呈固缩、凋亡样改变,神经细胞和神经毡内突触活性密度降低。拉莫三嗪组神经元胞膜轻微皱缩,局部少量水肿。愈痫灵组神经元核仁清晰,细胞内各结构清晰。提示愈痫灵颗粒、拉莫三嗪有拮抗KA致颞叶癫痫大鼠海马组织CA3区超微结构损伤的作用。3. Timm组织染色法观测海马CA3区MFS结果愈痫灵组、拉莫三嗪组苔藓纤维出芽MOD与IOD明显降低,两组与空白对照组、假手术组比较,MOD与IOD差异有统计学意义(P<0.05)；与模型组比较,MOD与IOD差异均具有统计学意义(P<0.05)；愈痫灵组与拉莫三嗪组比较,MOD与IOD差异均具有统计学意义(P<0.05),提示愈痫灵颗粒能显著降低KA致颞叶癫痫大鼠海马CA3区MFS总体阳性程度,且比拉莫三嗪改善其阳性程度的效果明显。4.免疫组化法检测苔藓纤维出芽标志物神经生长相关蛋白(GAP-43)结果愈痫灵组、拉莫三嗪组神经生长相关蛋白MOD与IOD明显降低,两组与空白对照组比较,MOD和IOD差异均有统计学意义(P<0.05)；与假手术组比较,差异无统计学意义(P>0.05)；与模型组比较,MOD和IOD差异均有显著统计学意义(P<0.01)；愈痫灵组与拉莫三嗪组比较,差异有有统计学意义(P<0.05),提示愈痫灵颗粒可抑制KA致颞叶癫痫大鼠海马CA3区GAP-43蛋白的表达,且疗效优于拉莫三嗪。[结论]1.KA致颞叶癫痫大鼠存在学习记忆能力下降。2.愈痫灵颗粒、拉莫三嗪可以改善KA致颞叶癫痫大鼠的学习记忆能力。3.愈痫灵颗粒改善KA致颞叶癫痫大鼠学习记忆能力的作用机制可能与其降低大鼠海马CA3区GAP-43免疫反应产物的过度表达,进而抑制海马苔藓纤维异常出芽,从而保护海马神经元有关。更多还原

【Abstract】 [Objective] To observe the effect of Yuxianling Granula on the learning and memory, hippocampus MFS(mossy fiber sprouting) of KA induced-temporal lobe epilepsy in rat,and to study the impact of the related mechanisms.[Methods] By stereotactic apparatus, we locally injected KA in hippocampus of rats,and established model of temporal lobe epilepsy, Induced seizures. To ev-aluate the effct of Yuxianling Granula on the learning and memory ability in th-e epileptic rats by the performance of learning and memory in rat water maze test; Using transmission electron microscopy to observe the changes of ultrastructural in hippocampal CA3 area; Using silver nitrate staining Timm to observe hippoca-mpal mossy fiber sprouting situation; To detect the change of the growth associa-ted protein (GAP43),which is markers of mossy fiber sprouting,by Immuno histo-chemical method,in order to exploer the effect of Yuxianling Granula on the lear-ning and memory,hippocampus MFS(mossy fiber sprouting) of KA induced tem-poral lobe epilepsy in rat,and to study the impact of the related mechanisms.[Results] 1.a water maze test resultsLearning and memory of Yuxianling Granula group and lamotrigine group improved significantly, compared with the control group, learning, memory per- formance was no significant difference (P> 0.05), compared with model group, learning and memory performance differences was statistically significant (P< 0.05); Comparation between Yuxianling Granula group and lamotrigine groups showed no significant difference (P> 0.05), it prompted that Yuxianling Granula, lamotrigine can both significantly improve learning and memory in epilepsy mo-del rats.2.Results of ultrastructure in hippocampal CA3 by TEMIn the control group, nuclei in CA3 neurons in hippocampus were round, m-embrane structure was clear, there were mitochondria, rough endoplasmic reticu-lum, smooth endoplasmic reticulum and lots of ribosomes in cytoplasm. In Sha-m group, we could see focal edema in the vicinity of glial cells. In Model group, the nuclear membrane was shrinkage, Axis reduced degeneration, Cytoplasmic o-rganelles is unclear, Heterochromatin increased margination obvious, borders we-re clearly. A wide range of nearby neurons was edema. Parts of glial cells was ed-ema obviously. Endothelial cell was shrinkage, apoptosis-like changes.Neurons and neuropil was within the lower density of synaptic activity. Lamotrigine group of neurons in the membrane slightly wrinkled, local small amount of edema. Nu-cleolus in Yuxianling Granula was clear, structure in the cell was clear. Tips Yu-xianling Granula, lamotrigine can antagonize hippocampus CA3 area ultrastr-ucture injury of KA induced temporal lobe epilepsy.3. Results of hippocampal CA3 area by Timm staining mossy fiber spr-outing MOD and IOD Yuxianling Granula group and lamotrigine group decreased obvioursly;Co-mpara with the control group and sham operation group,MOD and IOD was sign-ificantly (P<0.05); with model group, the difference between MOD and IOD was statistically significant (P<0.05); Comparation between Yuxianling Granula gro-up and the lamotrigine group, the differences between MOD and IOD were stat-istically significant (P< 0.05), It suggested that Yuxianling Granula can signiic-antly reduce the overall positive degree in hippocampal CA3 area MFS of KA i-nduced temporal lobe epilepsy, and the improving their positive effect in Yuxian ling Granula group was more clearly than that in Lamotriazine group.4. Results of Immunohistochemical detection of mossy fiber sprouting of nerve growth associated protein markers (GAP-43)Growth associated protein MOD and IOD in Yuxianling Granula group,La-motrigine group decreased, compared with the control group, differences betwen MOD and IOD in both group were statistically significant (P<0.05); compared with the sham group, the difference was not statistically significant (P> 0.05); co-mpared with model group, the differences were statistically signi-ficant (P<0.01); compared Yuxianling Granula group with the lamotrigine group, the difference w as statistically significant (P<0.05),It prompted Yuxianling Granula can inhibit t-he expression of the KA induced temporal lobe epilepsy rat hippocampal CA3 ar-ea GAP-43 protein, and more effectively than lamotrigine.[Conclusion]1. KA induced temporal lobe epilepsy rats can learn and memory. 2. Yuxianling Granula, lamotrigine can improve the learning and memory ability of KA induced temporal lobe epilepsy rat.3.The mechanism of Yuxianling Granula improving learning and memory of KA induced temporal lobe epilepsy rats may be related to its reduce in the over-Ex pression of hippocampal CA3 area GAP-43immune response product, there-by inhibiting hippocampal abnormal mossy fiber sprouting, and thus protect the hippocampal neurons.更多还原