本文選題：腦積水 + 海馬��； 參考：《新鄉(xiāng)醫(yī)學院》2014年碩士論文

【摘要】：目的建立腦積水(Subarachnoid hemorrhage) Sprague-Dawley (SD)大鼠模型,采用避暗試驗方法研究腦積水模型大鼠學習記憶功能,通過檢測神經(jīng)生長相關(guān)蛋白(neuronal growth associated protein, GAP-43)在腦積水模型大鼠海馬組織中表達,探討GAP-43與學習記憶之間的關(guān)系。 方法 1.腦積水Sprague-Dawley大鼠模型建立與學習記憶相關(guān)性研究：健康雄性SD大鼠160只,隨機分為腦積水模型組(n=96只),假手術(shù)組(n=32只),空白對照組(n=32只),腦積水模型組分1w,2w,4w,6w,共4個亞組,假手術(shù)組只進行手術(shù),注入生理鹽水,不注入白陶土懸液,空白對照組和模型組、假手術(shù)組飼養(yǎng)環(huán)境和喂食相同。觀察大鼠行為學改變,采用避暗試驗方法記錄每只大鼠實驗潛伏期(latentperiod.LT)以及來回穿梭次數(shù)(EN),研究學習記憶能力。 2.大鼠海馬GAP-43的表達與學習空間記憶的關(guān)系：各實驗組大鼠均采用單籠飼養(yǎng),飼養(yǎng)環(huán)境相同,避暗試驗檢測后,無痛處死腦積水模型組24只,假手術(shù)組8只及空白對照組大鼠8只,剩余大鼠進入下一亞組。腦積水模型組每亞組于1w,2w,4w,6w時行避暗實驗后無痛處死大鼠,模型組每亞組取大鼠n=24只,假手術(shù)組及空白對照組取n=8只；檢測GAP-43mRNA及蛋白表達表達。每模型實驗組大鼠2/3用乙醚麻醉斷頭,無RNA酶試驗臺內(nèi)快速取出海馬,并液氮冷藏后迅速移至-80℃冰箱備用,另1/3經(jīng)吸入乙醚后用4%多聚甲醛灌注、蔗糖梯度脫水后,應(yīng)用OCT(Sakura,USA)包埋固定,-80℃冰箱保存以備組織切片用。應(yīng)用免疫組化、RT-PCR和westernblot等技術(shù)方法研究腦積水模型大鼠海馬GAP-43mRNA及相關(guān)蛋白的表達并探討與學習及空間記憶的關(guān)系。 結(jié)果 1.腦積水模型大鼠制作成功,經(jīng)過動物MRI監(jiān)測系統(tǒng),確認為腦積水后,大鼠有認知改變表現(xiàn)。腦積水模型制作成功率為95.6%。腦積水模型組1w組避暗試驗結(jié)果的LT(51.73±3.62s)、EN為(3.89±±1.07次)；2w組避暗試驗結(jié)果為LT(44.51±1.28s)、EN為(4.35±1.19次)；4w組避暗試驗結(jié)果LT為(37.86±±3.57s)、EN為(5.71±0.49次)；6w組避暗試驗結(jié)果為(31.22±±3.18s)、EN為(3.96±±1.37次),假手術(shù)組避暗試驗結(jié)果LT為(53.144±3.11s)、EN為(3.55±±1.51次),空白對照組避暗試驗結(jié)果LT為(55.00±3.78s)、EN為(3.65±1.52次)。腦積水模型組與對照組比學習記憶能力明顯下降(P0.05),組間對比,模型組大鼠1w、2w、4w、6w組認知功能逐漸減弱,學習記憶有明顯減弱,有統(tǒng)計學意義(P0.05),均較空白組差,比值均有統(tǒng)計學意義(P0.05)。 2.分子生物學檢測顯示： (1)免疫組織化學結(jié)果：GAP-43蛋白為胞質(zhì)蛋白為棕黃色。陽性細胞百分率腦積水模型模型組與空白對照組、假手術(shù)組均有明顯差異性(P0.05),模型組明顯低于對照組。模型組組內(nèi)比較,陽性細胞百分率6w明顯低于1w組、2w組、4w組,6w組最低。隨時間延續(xù),腦受壓時間長,認知功能逐漸下降(P0.05)。 (2)RT-PCR反轉(zhuǎn)錄聚合酶鏈式反應(yīng)試驗結(jié)果：紫光燈下,naker的對照下目的條帶清晰可見,通過測定各條帶的光密度值校對值,腦積水模型組與對照組有明顯差異,模型組明顯低于對照組(P0.05)。模型組內(nèi)比較,光密度校對值模型組1w、2w、4w組明顯高于6w組,6w組最低(P0.05)。 (3)Vestern blot印記檢測結(jié)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maker的對照下,通過測定各目的條帶的光密度值,與管家基因蛋白比值校對,腦積水模型組與對照組有明顯差異(P0.05),模型組明顯低于對照組。模型組內(nèi)比較,光密度校對值模型組1w、2w、4w組時明顯高6w組,6w組最低(P0.05)。 結(jié)論 1.腦積水模型大鼠成功建立,腦積水模型大鼠學習記憶等認知功能受損。 2.腦積水模型大鼠海馬組織中GAP-43表達下調(diào),可能是學習記憶的重要分子機制之一。
[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.

【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】：R742.7

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