本文選題：高血脂　切入點(diǎn)：腦缺血　出處：《河北聯(lián)合大學(xué)》2014年碩士論文

【摘要】：目的探討跑臺(tái)運(yùn)動(dòng)對(duì)大鼠高血脂腦缺血再灌注后海馬區(qū)Ku蛋白和細(xì)胞周期蛋白的作用。 方法本實(shí)驗(yàn)將SPF級(jí)雄性高血脂誘導(dǎo)的Wistar大鼠120只隨機(jī)分為：對(duì)照組（n=40），模型組（n=40）、實(shí)驗(yàn)組（n=40）。高血脂模型采用高血脂飼料喂養(yǎng)6周形成高血脂大鼠后。實(shí)驗(yàn)組于建模前15天開(kāi)始進(jìn)行跑臺(tái)運(yùn)動(dòng)20m/min，30min/day，應(yīng)用四血管阻斷法（Pulsinelli4VO法）制備全腦缺血模型，分別于缺血15min再灌注后3h、6h、24h和48h四個(gè)時(shí)間點(diǎn)處死大鼠，缺血再灌注后24h和48h大鼠在處死前采用水迷宮檢測(cè)各組大鼠視空間記憶。各時(shí)間點(diǎn)的標(biāo)本采用蘇木素-伊紅染色法（hematoxylin-eosin staining，HE）觀察各組大鼠腦組織神經(jīng)元細(xì)胞形態(tài)的變化情況；采用免疫組織化學(xué)染色和免疫印跡蛋白（western-blotting）方法檢測(cè)各組大鼠腦組織中Ku70、Ku80、CycinA和CyclinE蛋白的表達(dá)情況。應(yīng)用SPSS17.0統(tǒng)計(jì)分析軟件對(duì)所得實(shí)驗(yàn)數(shù)據(jù)進(jìn)行重復(fù)設(shè)計(jì)的單因素方差分析，以P0.05，表示差異具有統(tǒng)計(jì)學(xué)意義。 結(jié)果1水迷宮檢測(cè)結(jié)果：與對(duì)照組比較，模型組大鼠在各時(shí)間點(diǎn)（24h、48h）的潛伏期時(shí)間顯著延長(zhǎng)，穿臺(tái)次數(shù)顯著減少，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；與模型組比較，實(shí)驗(yàn)組大鼠在各時(shí)間點(diǎn)（24h、48h）的潛伏期時(shí)間顯著縮短而穿臺(tái)次數(shù)顯著增加，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。2形態(tài)觀察結(jié)果（HE染色）對(duì)照組大鼠海馬區(qū)神經(jīng)元細(xì)胞排列整齊，胞漿淡染；模型組與對(duì)照組比較3h結(jié)構(gòu)形態(tài)即出現(xiàn)形態(tài)改變，表現(xiàn)為細(xì)胞失去正常結(jié)構(gòu)，部分細(xì)胞形態(tài)異常，6h海馬區(qū)可見(jiàn)較多腫脹的神經(jīng)元，間質(zhì)水腫，結(jié)構(gòu)疏松，神經(jīng)元和膠質(zhì)細(xì)胞出現(xiàn)核固縮、深染，也有部分神經(jīng)元胞核完全消失，形成空泡狀結(jié)構(gòu)；24h腦組織神經(jīng)細(xì)胞破壞，可見(jiàn)較多變性壞死的神經(jīng)細(xì)胞，神經(jīng)細(xì)胞呈空泡狀或海綿狀，神經(jīng)元細(xì)胞出現(xiàn)核固縮、碎裂和核仁消失現(xiàn)象，間質(zhì)水腫明顯；48h可見(jiàn)壞死神經(jīng)細(xì)胞，腦組織結(jié)構(gòu)破壞，結(jié)構(gòu)疏松，核膜碎裂、核仁消失，各時(shí)間點(diǎn)中以48h損傷最嚴(yán)重；實(shí)驗(yàn)組大鼠海馬區(qū)神經(jīng)元損傷較模型組明顯減輕，可見(jiàn)壞死的神經(jīng)元減少，間質(zhì)水腫較輕，核固縮和空泡較少。3Ku70免疫組化和Western blot分析結(jié)果：免疫組化結(jié)果：Ku70免疫組化染色呈棕黃色，定位于細(xì)胞胞漿中，主要表達(dá)在神經(jīng)元細(xì)胞。與對(duì)照組比較，模型組大鼠在各時(shí)間點(diǎn)（3h、6h、24h、48h）Ku70蛋白的表達(dá)增加，且24h達(dá)高峰，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；與模型組比較，實(shí)驗(yàn)組大鼠在各時(shí)間點(diǎn)（3h、6h、24h、48h）Ku70蛋白的表達(dá)明顯增加，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；Westernblot分析結(jié)果：與對(duì)照組比較，模型組大鼠在各時(shí)間點(diǎn)（3h、6h、24h、48h）Ku70蛋白的表達(dá)增加，且24h達(dá)高峰，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；與模型組比較，實(shí)驗(yàn)組大鼠在各時(shí)間點(diǎn)（3h、6h、24h、48h）Ku70蛋白的表達(dá)明顯增加，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。4Ku80免疫組化和Western blot分析結(jié)果：免疫組化結(jié)果：Ku80免疫組化染色呈棕黃色，定位于細(xì)胞胞核中，主要表達(dá)在海馬區(qū)神經(jīng)細(xì)胞。與對(duì)照組比較，模型組大鼠在各時(shí)間點(diǎn)（3h、6h、24h、48h）Ku80蛋白的表達(dá)明顯增加，且6h達(dá)高峰，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；與模型組比較，實(shí)驗(yàn)組大鼠在各時(shí)間點(diǎn)（3h、6h、24h、48h）Ku80蛋白的表達(dá)明顯增加，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；Western blot分析結(jié)果：與對(duì)照組比較，模型組大鼠在各時(shí)間點(diǎn)（3h、6h、24h、48h）Ku80蛋白的表達(dá)明顯增加，且6h達(dá)高峰，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；與模型組比較，實(shí)驗(yàn)組大鼠在各時(shí)間點(diǎn)（3h、6h、24h、48h）Ku80蛋白的表達(dá)明顯增加，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。5CyclinA免疫組化和Western blot分析結(jié)果：免疫組化結(jié)果：CyclinA免疫組化染色呈棕黃色，，定位于細(xì)胞胞核中，主要表達(dá)在海馬區(qū)神經(jīng)細(xì)胞。與對(duì)照組比較，模型組大鼠在各時(shí)間點(diǎn)（3h、6h、24h、48h）CyclinA蛋白的表達(dá)量逐漸增加，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；與模型組比較，實(shí)驗(yàn)組大鼠在各時(shí)間點(diǎn)（3h、6h、24h、48h）CyclinA蛋白的表達(dá)明顯增加，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；Western blot分析結(jié)果：與對(duì)照組比較，模型組大鼠在各時(shí)間點(diǎn)（3h、6h、24h、48h）CyclinA蛋白的表達(dá)量逐漸增加，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；與模型組比較，實(shí)驗(yàn)組大鼠在各時(shí)間點(diǎn)（3h、6h、24h、48h）CyclinA蛋白的表達(dá)明顯增加，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。6CyclinE免疫組化和Western blot分析結(jié)果：免疫組化結(jié)果：CyclinE免疫組化染色呈棕黃色，定位于細(xì)胞胞核中，主要表達(dá)在海馬區(qū)神經(jīng)細(xì)胞。與對(duì)照組比較，模型組大鼠在各時(shí)間點(diǎn)（3h、6h、24h、48h）CyclinE蛋白的表達(dá)逐漸降低，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；與模型組比較，實(shí)驗(yàn)組大鼠在各時(shí)間點(diǎn)（3h、6h、24h、48h）CyclinE蛋白的表達(dá)明顯增加，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；Western blot分析結(jié)果：與對(duì)照組比較，模型組大鼠在各時(shí)間點(diǎn)（3h、6h、24h、48h）CyclinE蛋白的表達(dá)降低，差異有統(tǒng)計(jì)學(xué)意義（P0.05）；與模型組比較，實(shí)驗(yàn)組大鼠在各時(shí)間點(diǎn)（3h、6h、24h、48h）CyclinE蛋白的表達(dá)明顯增加，差異有統(tǒng)計(jì)學(xué)意義（P0.05）。 結(jié)論1跑臺(tái)運(yùn)動(dòng)可改善全腦缺血再灌注大鼠空間記憶功能。2跑臺(tái)運(yùn)動(dòng)可增加Ku蛋白的表達(dá)，同時(shí)調(diào)控CyclinA和CyclinE的表達(dá)，對(duì)腦缺血有保護(hù)作用。
[Abstract]:Objective to investigate the effect of treadmill exercise on Ku protein and cyclin protein in hippocampus of rats after hyperlipidemia and cerebral ischemia reperfusion.
Methods the Wistar rats induced by SPF male hyperlipidemia 120 rats were randomly divided into control group (n=40), model group (n=40), experimental group (n=40). The model of hyperlipidemia with high lipid diet for 6 weeks after the formation of hyperlipemia rats. The experimental group began running 20m/min. In the 15 days before modeling 30min/day, using four vessel occlusion method (Pulsinelli4VO method) to prepare the model of whole brain ischemia in ischemic reperfusion after 15min of 3H, 6h, 24h and 48h four time points and the rats were killed after ischemia reperfusion 24h and 48h rats were sacrificed before using in water maze test in rats visuospatial memory. At different time points were detected by hematoxylin eosin staining (hematoxylin-eosin, staining, HE) to observe the morphological changes of brain tissue in rats of each group of neurons; using chemical staining and Western blot immunohistochemistry (Western-blotting) method to detect the rat brain The expression of Ku70, Ku80, CycinA and CyclinE protein in tissues was analyzed. SPSS17.0 analysis software was used to analyze the experimental data by one-way ANOVA, and the difference was statistically significant with P0.05.
The 1 water maze test results: compared with the control group, the rats in the model group at each time point (24h, 48h) the incubation time was prolonged and the frequency of crossing the platform was significantly reduced, the difference was statistically significant (P0.05); compared with the model group, the rats in the experimental group at each time point (24h, 48h). The latency time was significantly shortened and the times across the platform significantly increased, the difference was statistically significant (P0.05.2) the results of morphology (HE staining) in control neurons in hippocampus of rats in order, cytoplasmic staining; model group compared with the control group 3H morphology appeared morphological changes showed the cells lose their normal structure, abnormal part of the cell morphology, 6h in hippocampus neurons showed more swelling, interstitial edema, loose structure, nuclear pyknosis, appearance of neurons and glial cells stained, some neurons disappeared completely, the formation of air bubbles; 24h brain nerve cells There were lots of damage, degeneration and necrosis of nerve cells, nerve cells were vacuolated or cavernous, pyknosis neurons cells, fragmentation and nucleoli disappeared, interstitial edema obviously; 48h necrosis of nerve cells, brain structure damage, loose structure, nuclear fragmentation, nucleolus disappeared, at each time point in the most serious 48h damage; neurons in hippocampus of rats in experimental group injury significantly reduced compared with the model group, reduce the necrosis of neurons, interstitial edema, pyknosis and vacuolation less.3Ku70 immunohistochemistry and Western blot analysis results: immunohistochemical results: Ku70 immunohistochemical staining was brown, located in the cytoplasm that is mainly expressed in neuronal cells. Compared with the control group, the rats in the model group at each time point (3H, 6h, 24h, 48h) the expression of Ku70 protein increased, and reached the peak at 24h, the difference was statistically significant (P0.05); compared with the model group, 瀹為獙緇勫ぇ榧犲湪鍚勬椂闂寸偣(3h,6h,24h,48h)Ku70铔嬬櫧鐨勮〃杈炬槑鏄懼鍔

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