戊四氮点燃模型大鼠海马ATF3的表达变化与苔藓纤维出芽的关系

发布时间：2018-04-09 06:35

本文选题：转录激活因子3　切入点：苔藓纤维出芽　出处：《中南大学》2014年硕士论文

【摘要】：目的通过观察戊四氮(pentylenetetrazole; PTZ)大鼠点燃模型海马CA3区及齿状回区转录激活因子3(activating transcription factor3; ATF3)的动态表达变化,结合海马苔藓纤维出芽(mossy fiber sprouting; MFS)的程度,探讨其与MFS的关系及在癫痫过程中的作用。方法健康SD雄性成年大鼠150只,随机分为生理盐水对照组(60只)和PTZ实验组(90只)。实验组每日腹腔注射PTZ (30mg/kg)；同时生理盐水组注射等量生理盐水。按照首次腹腔注射后第3天,1周,2周,4周和6周的时间点分配,将PTZ组及生理盐水组随机分为5个亚组。根据实验方法的不同再将每个亚组分为三个小组：第一组用于免疫组化及免疫荧光,检测各时间点ATF3蛋白在海马各区的动态表达及在细胞内表达定位；第二组用于Western Blot,将海马各区ATF3表达变化进行半定量分析；第三组用于Timm染色,检测苔藓纤维出芽情况,其中实验组每小组各6只,对照组每小组各4只。结果 1.动物行为学表现大鼠多在连续注射PTZ21-28天(平均23.7±1.9天)后被点燃。模型点燃率为94.4%,死亡率为5.33%。除了4周和6周组各有一只大鼠未出现痫性发作外,点燃成功后大鼠出现诱发发作及自发发作,对照组未出现痫性发作。 2.苔藓纤维出芽评分实验组CA3区与对照组相对应时间点相比MFS评分均有显著性差异(P0.05)。从3天起评分逐渐增加,4周时达高峰,至6周仍维持在较高水平。齿状回区MFS评分在实验组与对照组之间以及各组内不同时间点之间比较均无显著性差异(P0.05)。 3.ATF3蛋白的动态表达免疫组化示在癫痫点燃过程中,实验组：海马CA3区ATF3蛋白表达逐渐上升,在4周组达最高峰,6周组稍有下降(P0.05)；齿状回区ATF3蛋白在点燃过程中无明显变化(P0.05)。对照组：海马CA3区及齿状回区在3天到6周组均无明显变化(P0.05)。Western Blot结果示海马ATF3的表达自3天组逐渐上升,至4周组达高峰,6周组较前明显下降；除了3天组之外,其他各时间点ATF3表达在实验组均较对照组高(P0.05)。免疫荧光结果示ATF3蛋白定位于大鼠海马神经元细胞核及细胞浆中,且主要集中在细胞浆。结论 ATF3蛋白在海马CA3区的表达上调可能促进苔藓纤维出芽,进而导致颞叶癫痫的发生及发展。
[Abstract]:Objective to observe the dynamic expression of 3(activating transcription factor 3 (ATF3) in the hippocampal CA3 and dentate gyrus of pentylenetetrazole (PTZ) kindling rat model, and to investigate the extent of mossy fiber sprouting in hippocampal mossy.To explore the relationship between MFS and Epilepsy and its role in epilepsy.Methods 150 healthy male SD rats were randomly divided into normal saline control group (n = 60) and PTZ experimental group (n = 90).The experimental group was intraperitoneally injected with 30 mg 路kg ~ (-1) of PTZ and the same amount of saline was injected into the saline group.According to the time points of 1 week, 2 weeks, 4 weeks and 6 weeks after the first intraperitoneal injection, the PTZ group and the saline group were randomly divided into 5 subgroups.Each subgroup was divided into three groups according to the different experimental methods. The first group was used for immunohistochemistry and immunofluorescence to detect the dynamic expression and localization of ATF3 protein in hippocampal regions at different time points.The second group was used for Western blot.The third group was used for Timm staining to detect the budding of moss fiber, 6 rats in each group in experimental group and 4 in control group.Result1.Animal behavior manifestationMost of the rats were kindled after continuous PTZ21-28 injection (mean 23.7 卤1.9 days).The model ignition rate was 94. 4% and the mortality was 5. 33%.Except for 4 weeks and 6 weeks group, there was no epileptic seizure in each group, but there was no epileptic attack in the control group after the success of kindling.2.Mossy fiber budding scoreCompared with the control group, the MFS scores in the CA3 area of the experimental group were significantly different from those in the control group (P 0.05).The score gradually increased from 3 days to reach the peak at 4 weeks and remained at a relatively high level at 6 weeks.There was no significant difference in MFS score of dentate gyrus between experimental group and control group and between groups at different time points (P 0.05).Dynamic expression of 3.ATF3 proteinImmunohistochemistry showed that the expression of ATF3 protein in the hippocampal CA3 region increased gradually during epileptic kindling, and decreased slightly in the 4th week group and the dentate gyrus region ATF3 protein in the 6th week group, while the ATF3 protein in the dentate gyrus area did not change significantly during the kindling process.Control group: there was no significant change in CA3 and dentate gyrus in hippocampus from 3 days to 6 weeks. The results of Western Blot showed that the expression of ATF3 in hippocampus increased gradually from 3 days to 6 weeks, and decreased significantly in the group of 4 weeks and 6 weeks, except for the group of 3 days.At other time points, the expression of ATF3 in the experimental group was higher than that in the control group (P 0.05).The results of immunofluorescence showed that the ATF3 protein was located in the nucleus and cytoplasm of hippocampal neurons of rats and mainly concentrated in the cytoplasm.ConclusionThe upregulation of ATF3 protein in hippocampal CA3 may promote mossy fiber sprouting, which may lead to the occurrence and development of temporal lobe epilepsy.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.1

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