本文選題：海人酸點燃癲癇 + 海馬　； 參考：《新鄉(xiāng)醫(yī)學院》2014年碩士論文

【摘要】：目的建立海人酸點燃癲癇大鼠模型,通過避暗試驗觀察海人酸點燃癲癇模型大鼠不同時間段學習記憶功能,檢測神經(jīng)生長相關蛋白(neuronal growth associated protein,GAP-43)在海人酸點燃癲癇模型大鼠海馬組織中的表達,探討GAP-43與學習記憶之間的關系。 方法 1.海人酸點燃癲癇大鼠模型的建立與學習記憶相關性研究：選6-8周齡健康雄性SD大鼠,體重220-250g。隨機將大鼠分為海人酸點燃癲癇模型1d組(模型1d組)、海人酸點燃癲癇模型7d組(模型7d組)、海人酸點燃癲癇模型30d組(模型30d組),假手術組和空白對照組(n=24)。模型1d組、模型7d組和模型30d組均用5μl微量注射器經(jīng)注藥套管給予1μl新鮮配置的海人酸(0.4mg·ml-1);假手術組同模型組手術方式,只注入生理鹽水,排除手術對大鼠學習記憶的影響；空白對照組,在每次刺激實驗組的大鼠時以相同的方法刺激抓取1-2次即可,以排除認為刺激對大鼠的影響。采用避暗試驗方法記錄各組大鼠實驗潛伏期(LT)及來回穿梭次數(shù)(EN),研究其學習及空間記憶功能。 2.大鼠海馬GAP-43的表達與學習空間記憶的關系：大鼠單籠飼養(yǎng),避暗試驗后,無外界刺激無痛處死。模型1d組、模型7d組和模型30d組避暗試驗后于1d,7d,30d時間點無痛處死大鼠,免疫組化、RT-PCR和Western blot技術檢測海馬組織中GAP-43蛋白及mRNA的表達。 結果 1.海人酸點燃癲癇大鼠模型的建立與學習記憶相關性研究結果：海人酸點燃癲癇模型大鼠制作成功后大鼠有認知改變表現(xiàn),檢測大鼠腦電波可收集典型的腦電波活動模型,模型制作成功率為95.6%。 模型1d組避暗試驗結果的LT(53.72±3.51s)、EN為(3.69±1.17次)；模型7d組避暗試驗結果為LT(44.51±1.28s)、EN為(4.15±1.18次)；模型30d組避暗試驗結果LT為(37.66±3.43s)、EN為(5.44±0.49次),假手術組避暗試驗結果LT為(53.14±3.11s)、EN為(3.55±1.51次),空白對照組避暗試驗結果LT為(55.33±3.28s)、EN為(3.65±1.52次)。與假手術組相比,模型7d組和模型30d組學習能力顯著降低(P0.05,P0.01)；模型組組間比較,模型1d、7d、30d組大鼠學習功能逐漸減弱,結果有統(tǒng)計學意義(P0.05,P0.01),假手術組與空白對照組無差異性。 2.分子生物學檢測顯示 (1)免疫組織化學結果：GAP-43免疫組織化學染色陽性結果表現(xiàn)為海馬細胞胞質(zhì)成棕黃色,集聚在細胞膜周圍。與假手術組相比,模型1d組、模型7d組和模型30d組GAP-43免疫陽性細胞數(shù)顯著降低(P0.05,P0.01)；與模型1d組相比,模型30d組GAP-43免疫陽性細胞數(shù)顯著降低(P0.01)。 (2)反轉錄聚合酶鏈式反應(Reverse Transcriptase Polymerase Chain Reaction, RT-PCR)結果：在Marker的對照下目的條帶清晰可見。通過測定各條帶的光密度值,與GADPH管家基因比值校對,與假手術組相比,模型1d組、模型7d組和模型30d組GAP-43mRNA表達顯著降低(P0.05)；與模型1d組、模型7d組相比,模型30d組GAP-43mRNA表達顯著降低(P0.01)；模型1d組與模型7d組間無顯著差異。 (3)免疫印跡分析(Western blot)結果：各個組大鼠海馬中GAP-43蛋白均有表達,在標準蛋白分子參照物Marker的對照下,通過測定各目的條帶的光密度值,與β-actin管家基因蛋白比值校對,與假手術組相比,模型1d組、模型7d組和模型30d組GAP-43蛋白表達顯著降低(P0.05,P0.01)；與模型1d組、模型7d組相比,模型30d組GAP-43蛋白表達顯著降低(P0.01)；與模型7d組相比,模型30d組GAP-43蛋白表達顯著降低(P0.01)。 結論 1.海人酸點燃癲癇可使大鼠學習記憶認知功能受損。 2.海人酸點燃癲癇大鼠海馬組織中GAP-43表達的下調(diào)可能是其認知功能受損的分子機制之一。
[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.
【學位授予單位】：新鄉(xiāng)醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R742.1

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本文編號：2115149