脑积水大鼠海马中GAP-43表达及其与学习记忆关系的研究

发布时间：2018-04-23 04:06

本文选题：脑积水 + 海马　；参考：《新乡医学院》2014年硕士论文

【摘要】：目的建立脑积水(Subarachnoid hemorrhage) Sprague-Dawley (SD)大鼠模型,采用避暗试验方法研究脑积水模型大鼠学习记忆功能,通过检测神经生长相关蛋白(neuronal growth associated protein, GAP-43)在脑积水模型大鼠海马组织中表达,探讨GAP-43与学习记忆之间的关系。方法 1.脑积水Sprague-Dawley大鼠模型建立与学习记忆相关性研究：健康雄性SD大鼠160只,随机分为脑积水模型组(n=96只),假手术组(n=32只),空白对照组(n=32只),脑积水模型组分1w,2w,4w,6w,共4个亚组,假手术组只进行手术,注入生理盐水,不注入白陶土悬液,空白对照组和模型组、假手术组饲养环境和喂食相同。观察大鼠行为学改变,采用避暗试验方法记录每只大鼠实验潜伏期(latentperiod.LT)以及来回穿梭次数(EN),研究学习记忆能力。 2.大鼠海马GAP-43的表达与学习空间记忆的关系：各实验组大鼠均采用单笼饲养,饲养环境相同,避暗试验检测后,无痛处死脑积水模型组24只,假手术组8只及空白对照组大鼠8只,剩余大鼠进入下一亚组。脑积水模型组每亚组于1w,2w,4w,6w时行避暗实验后无痛处死大鼠,模型组每亚组取大鼠n=24只,假手术组及空白对照组取n=8只；检测GAP-43mRNA及蛋白表达表达。每模型实验组大鼠2/3用乙醚麻醉断头,无RNA酶试验台内快速取出海马,并液氮冷藏后迅速移至-80℃冰箱备用,另1/3经吸入乙醚后用4%多聚甲醛灌注、蔗糖梯度脱水后,应用OCT(Sakura,USA)包埋固定,-80℃冰箱保存以备组织切片用。应用免疫组化、RT-PCR和westernblot等技术方法研究脑积水模型大鼠海马GAP-43mRNA及相关蛋白的表达并探讨与学习及空间记忆的关系。结果 1.脑积水模型大鼠制作成功,经过动物MRI监测系统,确认为脑积水后,大鼠有认知改变表现。脑积水模型制作成功率为95.6%。脑积水模型组1w组避暗试验结果的LT(51.73±3.62s)、EN为(3.89±±1.07次)；2w组避暗试验结果为LT(44.51±1.28s)、EN为(4.35±1.19次)；4w组避暗试验结果LT为(37.86±±3.57s)、EN为(5.71±0.49次)；6w组避暗试验结果为(31.22±±3.18s)、EN为(3.96±±1.37次),假手术组避暗试验结果LT为(53.144±3.11s)、EN为(3.55±±1.51次),空白对照组避暗试验结果LT为(55.00±3.78s)、EN为(3.65±1.52次)。脑积水模型组与对照组比学习记忆能力明显下降(P0.05),组间对比,模型组大鼠1w、2w、4w、6w组认知功能逐渐减弱,学习记忆有明显减弱,有统计学意义(P0.05),均较空白组差,比值均有统计学意义(P0.05)。 2.分子生物学检测显示： (1)免疫组织化学结果：GAP-43蛋白为胞质蛋白为棕黄色。阳性细胞百分率脑积水模型模型组与空白对照组、假手术组均有明显差异性(P0.05),模型组明显低于对照组。模型组组内比较,阳性细胞百分率6w明显低于1w组、2w组、4w组,6w组最低。随时间延续,脑受压时间长,认知功能逐渐下降(P0.05)。 (2)RT-PCR反转录聚合酶链式反应试验结果：紫光灯下,naker的对照下目的条带清晰可见,通过测定各条带的光密度值校对值,脑积水模型组与对照组有明显差异,模型组明显低于对照组(P0.05)。模型组内比较,光密度校对值模型组1w、2w、4w组明显高于6w组,6w组最低(P0.05)。 (3)Vestern blot印记检测结果：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maker的对照下,通过测定各目的条带的光密度值,与管家基因蛋白比值校对,脑积水模型组与对照组有明显差异(P0.05),模型组明显低于对照组。模型组内比较,光密度校对值模型组1w、2w、4w组时明显高6w组,6w组最低(P0.05)。结论 1.脑积水模型大鼠成功建立,脑积水模型大鼠学习记忆等认知功能受损。 2.脑积水模型大鼠海马组织中GAP-43表达下调,可能是学习记忆的重要分子机制之一。
[Abstract]:Objective to establish the rat model of Subarachnoid hemorrhage Sprague-Dawley (SD), and to study the learning and memory function of the rat model of hydrocephalus by dark test, and to investigate the expression of neuronal growth associated protein (GAP-43) in the hippocampus of the rat model of hydrocephalus, and explore the GAP-43 and learning records. The relationship between memories.
Method
1. Sprague-Dawley rat model of hydrocephalus and learning and memory correlation: 160 healthy male SD rats were randomly divided into the hydrocephalus model group (n=96 only), the sham operation group (n=32), the blank control group (n=32 only), the hydrocephalus model group of 1W, 2W, 4W, 6W, and the sham operation group was only operated, injected physiological saline, and did not infuse white pottery. Soil suspension, blank control group and model group, sham operation group feeding the same environment and feeding. Observe the behavior changes of rats, use the dark test method to record the incubation period of each rat (latentperiod.LT) and the number of shuttle back and forth (EN), and study the learning and memory ability.
The relationship between the expression of GAP-43 in the hippocampus of 2. rats and the learning space memory: the rats in the experimental group were fed with single cage, and the environment was the same. After the dark test, 24 rats were killed without pain, 8 rats in the sham operation group and 8 rats in the blank control group, and the remaining rats entered the next subgroup. Each subgroup of the hydrocephalus group was in the 1W, 2W, 4W, 6W. Rats were killed without pain after dark experiment. The rats in each group of the model group were taken n=24 only, the sham operation group and the blank control group were taken n=8 only, and the expression of GAP-43mRNA and protein were detected. The 2/3 of each model experimental group was anaesthetized with ether, and the hippocampus was quickly removed from the RNA enzyme test platform, and the liquid nitrogen was quickly moved to -80 centigrade refrigerators after cold storage, and 1/3 classics. After the inhalation of ether, 4% polyoxymethylene was perfused. After the sucrose was dehydrated, OCT (Sakura, USA) was embedded and fixed, and -80 C refrigerator was preserved for tissue section. The expression of GAP-43mRNA and related proteins in hippocampus of rat model of hydrocephalus was studied by immunohistochemistry, RT-PCR and Westernblot. The relationship between learning and spatial memory was discussed.
Result
1. rat model of hydrocephalus was made successfully. After the animal MRI monitoring system, it was confirmed that the rat had cognitive change after hydrocephalus. The success rate of the hydrocephalus model was LT (51.73 + 3.62s) and EN (3.89 + 1.07 times) in the 1W group of the model group of hydrocephalus, EN (3.89 + 1.07 times), and LT (44.51 + 1.28s) and EN (4.35 + 1.19) in the 2W group. The results of 4W group were (37.86 + + 3.57s) and EN (5.71 + 0.49 times); 6W group dark test results were (31.22 + + 3.18s), EN was (3.96 + 1.37), LT was (53.144 + 3.11s), EN was (3.55 + + 1.51) and LT was (55 + 3.78s) in empty white control group (3.65 + 1.52). Hydrocephalus was (3.65 + 1.52). The learning and memory ability of the model group and the control group decreased significantly (P0.05). The cognitive function of the rats in the model group 1W, 2W, 4W, 6W was gradually weakened, and the learning and memory decreased significantly (P0.05), all of which were worse than those in the blank group (P0.05).
2. molecular biological tests showed that:
(1) immunohistochemical results: GAP-43 protein was a brown yellow cytoplasmic protein. The percentage of positive cells in the hydrocephalus model group and the blank control group were significantly different (P0.05), and the model group was significantly lower than the control group. The percentage of positive cells in the model group was significantly lower than that of the 1W group, the 2W group, the 4W group, and the 6W group were the lowest. The duration of brain compression was longer, and cognitive function gradually decreased (P0.05).
(2) RT-PCR reverse transcriptional polymerase chain reaction test results: under the purple light, the target band of naker was clearly visible, by measuring the value of the light density value of each band, the hydrocephalus model group was significantly different from the control group, the model group was significantly lower than the control group (P0.05). The comparison of the model group, the light density proofreading group 1W, 2W, 4W group Significantly higher than the 6W group, the 6W group was the lowest (P0.05).
(3) the results of Vestern blot imprint detection: the GAP-43 protein in the hippocampus of each group was expressed. Under the control of the standard protein reference substance maker, the value of light density of the target bands was measured and the ratio of the gene protein of the housekeeper was proofed. The model group of the hydrocephalus was significantly different from the control group (P0.05). The model group was significantly lower than the control group. Compared with the model group, the optical density calibration model group 1W, 2W, 4W group was significantly higher than the 6W group, while the 6W group was the lowest (P0.05).
conclusion
1. rat models of hydrocephalus were successfully established, and rats with hydrocephalus were impaired in learning, memory and other cognitive functions.
2. downregulation of GAP-43 expression in hippocampal tissues of rats with hydrocephalus may be one of the important molecular mechanisms of learning and memory.

【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】：R742.7

【参考文献】