海人酸点燃癫痫大鼠海马GAP-43的表达及其与学习记忆的关系

发布时间：2018-07-11 12:10

  本文选题：海人酸点燃癫痫 + 海马　； 参考：《新乡医学院》2014年硕士论文

【摘要】：目的建立海人酸点燃癫痫大鼠模型,通过避暗试验观察海人酸点燃癫痫模型大鼠不同时间段学习记忆功能,检测神经生长相关蛋白(neuronal growth associated protein,GAP-43)在海人酸点燃癫痫模型大鼠海马组织中的表达,探讨GAP-43与学习记忆之间的关系。 方法 1.海人酸点燃癫痫大鼠模型的建立与学习记忆相关性研究：选6-8周龄健康雄性SD大鼠,体重220-250g。随机将大鼠分为海人酸点燃癫痫模型1d组(模型1d组)、海人酸点燃癫痫模型7d组(模型7d组)、海人酸点燃癫痫模型30d组(模型30d组),假手术组和空白对照组(n=24)。模型1d组、模型7d组和模型30d组均用5μl微量注射器经注药套管给予1μl新鲜配置的海人酸(0.4mg·ml-1);假手术组同模型组手术方式,只注入生理盐水,排除手术对大鼠学习记忆的影响；空白对照组,在每次刺激实验组的大鼠时以相同的方法刺激抓取1-2次即可,以排除认为刺激对大鼠的影响。采用避暗试验方法记录各组大鼠实验潜伏期(LT)及来回穿梭次数(EN),研究其学习及空间记忆功能。 2.大鼠海马GAP-43的表达与学习空间记忆的关系：大鼠单笼饲养,避暗试验后,无外界刺激无痛处死。模型1d组、模型7d组和模型30d组避暗试验后于1d,7d,30d时间点无痛处死大鼠,免疫组化、RT-PCR和Western blot技术检测海马组织中GAP-43蛋白及mRNA的表达。 结果 1.海人酸点燃癫痫大鼠模型的建立与学习记忆相关性研究结果：海人酸点燃癫痫模型大鼠制作成功后大鼠有认知改变表现,检测大鼠脑电波可收集典型的脑电波活动模型,模型制作成功率为95.6%。 模型1d组避暗试验结果的LT(53.72±3.51s)、EN为(3.69±1.17次)；模型7d组避暗试验结果为LT(44.51±1.28s)、EN为(4.15±1.18次)；模型30d组避暗试验结果LT为(37.66±3.43s)、EN为(5.44±0.49次),假手术组避暗试验结果LT为(53.14±3.11s)、EN为(3.55±1.51次),空白对照组避暗试验结果LT为(55.33±3.28s)、EN为(3.65±1.52次)。与假手术组相比,模型7d组和模型30d组学习能力显著降低(P0.05,P0.01)；模型组组间比较,模型1d、7d、30d组大鼠学习功能逐渐减弱,结果有统计学意义(P0.05,P0.01),假手术组与空白对照组无差异性。 2.分子生物学检测显示 (1)免疫组织化学结果：GAP-43免疫组织化学染色阳性结果表现为海马细胞胞质成棕黄色,集聚在细胞膜周围。与假手术组相比,模型1d组、模型7d组和模型30d组GAP-43免疫阳性细胞数显著降低(P0.05,P0.01)；与模型1d组相比,模型30d组GAP-43免疫阳性细胞数显著降低(P0.01)。 (2)反转录聚合酶链式反应(Reverse Transcriptase Polymerase Chain Reaction, RT-PCR)结果：在Marker的对照下目的条带清晰可见。通过测定各条带的光密度值,与GADPH管家基因比值校对,与假手术组相比,模型1d组、模型7d组和模型30d组GAP-43mRNA表达显著降低(P0.05)；与模型1d组、模型7d组相比,模型30d组GAP-43mRNA表达显著降低(P0.01)；模型1d组与模型7d组间无显著差异。 (3)免疫印迹分析(Western blot)结果：各个组大鼠海马中GAP-43蛋白均有表达,在标准蛋白分子参照物Marker的对照下,通过测定各目的条带的光密度值,与β-actin管家基因蛋白比值校对,与假手术组相比,模型1d组、模型7d组和模型30d组GAP-43蛋白表达显著降低(P0.05,P0.01)；与模型1d组、模型7d组相比,模型30d组GAP-43蛋白表达显著降低(P0.01)；与模型7d组相比,模型30d组GAP-43蛋白表达显著降低(P0.01)。 结论 1.海人酸点燃癫痫可使大鼠学习记忆认知功能受损。 2.海人酸点燃癫痫大鼠海马组织中GAP-43表达的下调可能是其认知功能受损的分子机制之一。
[Abstract]:Objective to establish an epileptic rat model of sea human acid kindling, and to observe the learning and memory function of the rat model of neuronal growth associated protein (GAP-43) in the hippocampus of epileptic rat model of sea human acid kindled model by dark test and observe the expression of GAP-43 and learning memory in the hippocampus of the rat model of sea human acid kindled epilepsy model. The relationship between them.
Method
Study on the relationship between the establishment of 1. sea human acid kindled epileptic rats model and learning memory correlation: 6-8 weeks old healthy male SD rats were selected. The weight 220-250g. randomly divided rats into 1D group (model 1D group), 7d group (model 7D), 30d group (model 30d) and sham operation group of sea human acid kindled epilepsy model (model 30d group), sham operation group. And blank control group (n=24). Model 1D group, model 7d group and model 30d group were treated with 1 u l fresh collocated sea human acid (0.4mg ML-1) with 5 u l micro injector via injection cannula. The operation mode of sham operation group and model group only injected physiological saline, and the effect of operation on learning and memory of rats was excluded. Blank control group was used in each stimulation experimental group. In rats, the rats were captured 1-2 times by the same method to eliminate the effect of stimulation on rats. The incubation period (LT) and the frequency of shuttle back and forth (EN) were recorded by dark test. The learning and spatial memory function of the rats were studied.
The relationship between the expression of GAP-43 in the hippocampus of 2. rats and learning space memory: rats were fed in a single cage and no external stimuli were painless after the dark test. Model 1D group, model 7d group and model 30d group were painless at 1D, 7d, 30d points after dark test, immunohistochemistry, RT-PCR and Western blot were used to detect GAP-43 protein and mRNA in the hippocampus Expression.
Result
The relationship between the establishment of the model of 1. sea human acid kindling epileptic rat and the study of learning and memory: the rat model of brain waves can be collected and the model of brain wave activity can be collected. The success rate of the model is 95.6%..
The results of the model 1D group were LT (53.72 + 3.51s) and EN (3.69 + 1.17 times); the result of 7D group in the 7d group was LT (44.51 + 1.28s) and EN (4.15 + 1.18); LT in the model 30d group was (37.66 + 0.49), EN was (53.14 + 0.49), and (3.55 + 1.51), blank control The results of group LT were (55.33 + 3.28s) and EN (3.65 + 1.52 times). Compared with sham operation group, the learning ability of model 7d group and model 30d group decreased significantly (P0.05, P0.01). The learning function of model 1D, 7d, 30d group gradually weakened, and the results were statistically significant (P0.05, P0.01), and there was no difference between the sham operation group and the blank control group. Sex.
2. molecular biological detection display
(1) immunohistochemical results: the positive results of GAP-43 immunohistochemical staining showed that the cytoplasm of the hippocampus was brown and yellow and gathered around the cell membrane. Compared with the sham group, the number of GAP-43 immunoreactive cells in the model 1D group, the model 7d group and the model 30d group decreased significantly (P0.05, P0.01), and the 30d group GAP-43 immunization of the model 1D group was compared with the model 1D group. The number of positive cells decreased significantly (P0.01).
(2) results of reverse transcriptional polymerase chain reaction (Reverse Transcriptase Polymerase Chain Reaction, RT-PCR): the target strip was clearly visible under the control of Marker. By measuring the light density of each band and proofreading the ratio of the GADPH housekeeper gene, the GAP-43mRNA expression was significant compared with the sham group, the model 1D group, the model 7d group and the model 30d group. Decrease (P0.05); compared with model 1D group and model 7d group, the expression of GAP-43mRNA in model group 30d was significantly decreased (P0.01); there was no significant difference between model 1D group and model 7d group.
(3) the results of immunoblotting analysis (Western blot): the GAP-43 protein in the hippocampus of each group was expressed. Under the control of the standard protein molecular reference Marker, the light density value of the target bands was measured and the ratio of the gene protein ratio of the beta -actin housekeeper was proofed. Compared with the sham operation group, the model 1D group, the model 7d group and the model 30d group GAP-43 protein. The expression of GAP-43 protein was significantly reduced (P0.05, P0.01). Compared with model 1D group and model 7d group, the expression of GAP-43 protein in the model 30d group decreased significantly (P0.01), and the expression of GAP-43 protein in the model 30d group was significantly lower than that in the model 7d group (P0.01).
conclusion
1. kainic acid kindled epilepsy can damage the learning and memory function of rats.
2. the downregulation of GAP-43 expression in hippocampus of kainic acid kindled epileptic rats may be one of the molecular mechanisms of cognitive impairment.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.1

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