跑台运动对脑缺血大鼠空间记忆、Ku蛋白与细胞周期蛋白的影响

发布时间：2018-04-01 02:02

  本文选题：高血脂　切入点：脑缺血　出处：《河北联合大学》2014年硕士论文

【摘要】：目的探讨跑台运动对大鼠高血脂脑缺血再灌注后海马区Ku蛋白和细胞周期蛋白的作用。 方法本实验将SPF级雄性高血脂诱导的Wistar大鼠120只随机分为：对照组（n=40），模型组（n=40）、实验组（n=40）。高血脂模型采用高血脂饲料喂养6周形成高血脂大鼠后。实验组于建模前15天开始进行跑台运动20m/min，30min/day，应用四血管阻断法（Pulsinelli4VO法）制备全脑缺血模型，分别于缺血15min再灌注后3h、6h、24h和48h四个时间点处死大鼠，缺血再灌注后24h和48h大鼠在处死前采用水迷宫检测各组大鼠视空间记忆。各时间点的标本采用苏木素-伊红染色法（hematoxylin-eosin staining，HE）观察各组大鼠脑组织神经元细胞形态的变化情况；采用免疫组织化学染色和免疫印迹蛋白（western-blotting）方法检测各组大鼠脑组织中Ku70、Ku80、CycinA和CyclinE蛋白的表达情况。应用SPSS17.0统计分析软件对所得实验数据进行重复设计的单因素方差分析，以P0.05，表示差异具有统计学意义。 结果1水迷宫检测结果：与对照组比较，模型组大鼠在各时间点（24h、48h）的潜伏期时间显著延长，穿台次数显著减少，差异有统计学意义（P0.05）；与模型组比较，实验组大鼠在各时间点（24h、48h）的潜伏期时间显著缩短而穿台次数显著增加，差异有统计学意义（P0.05）。2形态观察结果（HE染色）对照组大鼠海马区神经元细胞排列整齐，胞浆淡染；模型组与对照组比较3h结构形态即出现形态改变，表现为细胞失去正常结构，部分细胞形态异常，6h海马区可见较多肿胀的神经元，间质水肿，结构疏松，神经元和胶质细胞出现核固缩、深染，也有部分神经元胞核完全消失，形成空泡状结构；24h脑组织神经细胞破坏，可见较多变性坏死的神经细胞，神经细胞呈空泡状或海绵状，神经元细胞出现核固缩、碎裂和核仁消失现象，间质水肿明显；48h可见坏死神经细胞，脑组织结构破坏，结构疏松，核膜碎裂、核仁消失，各时间点中以48h损伤最严重；实验组大鼠海马区神经元损伤较模型组明显减轻，可见坏死的神经元减少，间质水肿较轻，核固缩和空泡较少。3Ku70免疫组化和Western blot分析结果：免疫组化结果：Ku70免疫组化染色呈棕黄色，定位于细胞胞浆中，主要表达在神经元细胞。与对照组比较，模型组大鼠在各时间点（3h、6h、24h、48h）Ku70蛋白的表达增加，且24h达高峰，差异有统计学意义（P0.05）；与模型组比较，实验组大鼠在各时间点（3h、6h、24h、48h）Ku70蛋白的表达明显增加，差异有统计学意义（P0.05）；Westernblot分析结果：与对照组比较，模型组大鼠在各时间点（3h、6h、24h、48h）Ku70蛋白的表达增加，且24h达高峰，差异有统计学意义（P0.05）；与模型组比较，实验组大鼠在各时间点（3h、6h、24h、48h）Ku70蛋白的表达明显增加，差异有统计学意义（P0.05）。4Ku80免疫组化和Western blot分析结果：免疫组化结果：Ku80免疫组化染色呈棕黄色，定位于细胞胞核中，主要表达在海马区神经细胞。与对照组比较，模型组大鼠在各时间点（3h、6h、24h、48h）Ku80蛋白的表达明显增加，且6h达高峰，差异有统计学意义（P0.05）；与模型组比较，实验组大鼠在各时间点（3h、6h、24h、48h）Ku80蛋白的表达明显增加，差异有统计学意义（P0.05）；Western blot分析结果：与对照组比较，模型组大鼠在各时间点（3h、6h、24h、48h）Ku80蛋白的表达明显增加，且6h达高峰，差异有统计学意义（P0.05）；与模型组比较，实验组大鼠在各时间点（3h、6h、24h、48h）Ku80蛋白的表达明显增加，差异有统计学意义（P0.05）。5CyclinA免疫组化和Western blot分析结果：免疫组化结果：CyclinA免疫组化染色呈棕黄色，，定位于细胞胞核中，主要表达在海马区神经细胞。与对照组比较，模型组大鼠在各时间点（3h、6h、24h、48h）CyclinA蛋白的表达量逐渐增加，差异有统计学意义（P0.05）；与模型组比较，实验组大鼠在各时间点（3h、6h、24h、48h）CyclinA蛋白的表达明显增加，差异有统计学意义（P0.05）；Western blot分析结果：与对照组比较，模型组大鼠在各时间点（3h、6h、24h、48h）CyclinA蛋白的表达量逐渐增加，差异有统计学意义（P0.05）；与模型组比较，实验组大鼠在各时间点（3h、6h、24h、48h）CyclinA蛋白的表达明显增加，差异有统计学意义（P0.05）。6CyclinE免疫组化和Western blot分析结果：免疫组化结果：CyclinE免疫组化染色呈棕黄色，定位于细胞胞核中，主要表达在海马区神经细胞。与对照组比较，模型组大鼠在各时间点（3h、6h、24h、48h）CyclinE蛋白的表达逐渐降低，差异有统计学意义（P0.05）；与模型组比较，实验组大鼠在各时间点（3h、6h、24h、48h）CyclinE蛋白的表达明显增加，差异有统计学意义（P0.05）；Western blot分析结果：与对照组比较，模型组大鼠在各时间点（3h、6h、24h、48h）CyclinE蛋白的表达降低，差异有统计学意义（P0.05）；与模型组比较，实验组大鼠在各时间点（3h、6h、24h、48h）CyclinE蛋白的表达明显增加，差异有统计学意义（P0.05）。 结论1跑台运动可改善全脑缺血再灌注大鼠空间记忆功能。2跑台运动可增加Ku蛋白的表达，同时调控CyclinA和CyclinE的表达，对脑缺血有保护作用。
[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, 瀹為獙缁勫ぇ榧犲湪鍚勬椂闂寸偣(3h,6h,24h,48h)Ku70铔嬬櫧鐨勮〃杈炬槑鏄惧�炲�

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