卵巢切除对大鼠空间记忆的影响及其和海马胰岛素信号相关性的研究

发布时间：2018-04-17 22:30

本文选题：雌激素 + 胰岛素　；参考：《兰州大学》2014年硕士论文

【摘要】：目的：更年期综合症是指女性在围绝经期因雌激素水平下降引起的以自主神经功能紊乱为主的一组症候群。临床发现更年期综合症患者同时伴有记忆功能下降。动物研究用卵巢去势鼠建立手术更年期模型,通过Morris水迷宫和恐惧条件反射等行为学测试,发现卵巢去势引起的雌激素水平低下可明显损害空间学习记忆；卵巢去势鼠用雌激素替代治疗,可增强海马CA1-CA3区的树突棘密度和LTP。雌激素和学习记忆的关系虽然有大量报道,但雌激素参与脑内信息处理的具体机制仍不明确。此外,神经系统高效利用能量的过程是葡萄糖的有氧氧化,学习记忆过程伴随着特定脑区葡萄糖利用率的增加,海马是学习记忆的关键脑区,基于任务模式的学习行为可见海马区葡萄糖摄取增加。细胞对葡萄糖的摄取借助于细胞膜上特定的葡萄糖转运体实现,葡萄糖转运体有多个亚型,其中GLUT4在骨骼肌及脂肪组织分布广泛,且具有胰岛素依赖性的特点。近年发现海马神经细胞有GLUT4的表达,这表明海马神经细胞的葡萄糖利用受胰岛素信号的调节。而胰岛素信号及胰岛素敏感性受多种信号的影响,有研究观察到雌激素可增强胰岛素敏感性,同时发现雌激素可以调控胰岛素的水平。雌激素对胰岛素的这种调节作用是否参与海马对信息处理的过程尚不得而知。因此,本研究我们通过大鼠卵巢去势构建雌激素水平低下模型,观察对海马空间记忆的影响及其与脑内胰岛素信号的关系。方法：动物处理：SD雌鼠(体重250g左右)随机分为四组：卵巢去势组(OVX)、正常组(Normal)、他莫昔芬组(N+TAM)以及抗胰岛素组(N+Anti)。对卵巢去势组进行双侧卵巢切除,正常组、他莫昔芬组和抗胰岛素组进行假手术(除不进行卵巢切除,其余各步骤均与卵巢去势组相同),术后恢复7天,各组利用脑立体定位仪进行双侧海马置管(bregma向后3.60mm,正中线两侧2.00mm,深度3.50mm)。海马置管后恢复7天,即进入正式实验。他莫昔芬组行为学连续训练5天,每天训练后立即给予腹腔注射10mg/kg他莫昔芬,用来阻断雌激素受体β (ERβ),胰岛素抗体组每天训练后立即给予双侧海马内注射胰岛素抗体(2μl/side)。各组大鼠于行为学检测前6h给予海马注射2mMol的2-NBDG(2μl/side)。行为学测试后立即处死。取材：各组动物处死前先采集脑脊液备用,后经心脏采血,分离血清备用。各组动物中一部分动物(每组6只动物)经灌注、固定、连续冰冻切片,保存备用。各组动物中另外一部分动物(每组6只)立即断头取脑,分离双侧海马,液氮冷冻备用。检测：行为学检测,利用智能水迷宫系统,检测大鼠学习记忆功能的变化。生化检测,利用ELISA技术检测血清,脑脊液及海马组织中胰岛素水平；在脑的连续冠状切片上,选取海马平面(Bregma -3.30mm,-3.45mm,-3.60mm,-3.75mm,-3.90mm,-4.05mm),利用荧光显微镜观察2-NBDG在海马区的分布,并计数海马CA3区2-NBDG阳性细胞的数量；提取海马组织的总RNA,利用Q-RT-PCR技术检测海马组织胰岛素mRNA的表达水平；提取海马组织总蛋白,利用Western blotting检测ERβ和GLUT4的蛋白表达水平。结果：1.卵巢去势降低大鼠空间学习记忆能力：卵巢去势后SD雌鼠出现明显的空间学习记忆障碍；同时,卵巢去势组与正常组相比,海马葡萄糖摄取显著降低,海马GLUT4蛋白表达水平明显下降;海马胰岛素的mRNA和蛋白表达水平均降低。2.卵巢去势对空间学习记忆的影响和雌激素受体p受体有关：他莫昔芬阻断ERp后,大鼠空间学习记忆出现明显损害；同时,他莫昔芬组的海马葡萄糖摄取下降,海马GLUT4蛋白表达水平降低,与正常组比；他莫昔芬组与正常组相比,海马胰岛素的mRNA和蛋白表达水平均下降。3.卵巢去势对空间学习记忆的影响和海马区胰岛素水平及葡萄糖利用有关：胰岛素抗体中和海马胰岛素后,与正常组相比,空间学习记忆明显损害,海马葡萄糖摄取及海马GLUT4蛋白表达水平显著下降。结论：雌激素对海马依赖性空间学习记忆的作用是通过雌激素受体p (ERβ)完成的：雌激素影响空间学习记忆的过程中有海马胰岛素信号的参与；在空间学习记忆过程中,雌激素通过影响海马胰岛素水平从而降低海马葡萄糖代谢。
[Abstract]:Objective: menopause syndrome refers to women in the perimenopausal period due to a drop in estrogen levels caused by autonomic nerve function disorder a syndrome. The clinical findings of climacteric syndrome were associated with memory decline. The establishment of animal research model surgery more years in ovariectomized rats, tested by Morris water maze and fear conditioning behavior found, ovariectomy caused by low estrogen levels can damage the spatial learning and memory in ovariectomized rats; estrogen replacement therapy, can enhance the relationship between the hippocampal CA1-CA3 region of dendritic spines and LTP. female hormone and learning and memory, although there are a lot of reports, but the specific mechanism of estrogen is involved in information processing in the brain is still not clear. In addition, nerve the efficient use of energy system is the process of aerobic oxidation of glucose, the process of learning and memory with glucose utilization in specific regions of the brain The increase of the hippocampus is the key brain area for learning and memory tasks increase learning behavior of glucose in hippocampus in uptake based on the uptake of glucose into cells by cell membrane specific glucose transporter, glucose transporter has multiple subtypes, of which GLUT4 is widely distributed in skeletal muscle and adipose tissue, and has the characteristics of insulin the dependence of the expression of hippocampal nerve cells. In recent years GLUT4, suggesting that the hippocampal neural cells by glucose insulin signal. The insulin signaling and insulin sensitivity is affected by various signals, studies have observed that estrogen enhances insulin sensitivity, also found that estrogen can regulate insulin levels. Whether this kind of estrogen on insulin the role of information processing in the hippocampus still can make nothing of it. Therefore, in this study we by rats Construction of the low level of estrogen in ovariectomized model, observe the relationship between effects on hippocampal spatial memory and brain insulin signaling. Methods: Animal: female SD rats (weight 250g) were randomly divided into four groups: ovariectomy group (OVX), normal group (Normal), he (N+TAM) and tamoxifen group insulin resistance group (N+Anti). The ovariectomized group were ovariectomized, normal group, tamoxifen group and insulin resistance group (except the sham operation without ovariectomy, the rest of the steps are the same with the ovariectomized group), postoperative recovery after 7 days, each group of bilateral hippocampal catheter by stereotaxic instrument (bregma back 3.60mm, 2.00mm on both sides of the midline, the depth of 3.50mm). The hippocampus catheter after 7 days of recovery, namely into the formal experiment. The tamoxifen group behavior for 5 days of training, given intraperitoneal injection of 10mg/kg immediately after tamoxifen training every day, to block estrogen Hormone receptor beta (ER beta), bilateral intrahippocampal injection of insulin antibody immediately after training daily insulin antibody group (2 l/side). The rats in the behavioral test at 6h prior to 2mMol injection into the hippocampus of 2-NBDG (2 l/side) were killed. Immediately after the behavioral test. Materials: first acquisition of cerebrospinal fluid reserve in each group from before death, after the heart blood serum was isolated. A part of each animal in animal (6 in each animal) by perfusion, fixed, continuous frozen sections of each animal preservation reserve. The other part of the animal (n = 6) immediately decapitated, bilateral hippocampus, frozen in liquid nitrogen detection: standby. Behavioral testing, the use of intelligent water maze system, to detect the changes of learning and memory function in rats. Serum biochemical testing, using ELISA technology, the level of insulin in the hippocampus and cerebrospinal fluid; continuous coronal sections of brain hippocampus on the selected plane (B Regma -3.30mm, -3.45mm, -3.60mm, -3.75mm, -3.90mm, -4.05mm), the distribution of 2-NBDG by fluorescence microscopy in the hippocampus, and count the number of 2-NBDG positive cells in hippocampal CA3 area; extraction of total RNA in the hippocampus, the expression of insulin mRNA in the hippocampus of Q-RT-PCR technology; extraction of total protein in the hippocampus, the use of Western blotting detection of ER beta and GLUT4 protein expression levels. Results: 1. ovariectomized rats decreased the ability of spatial learning and memory: ovariectomized female SD rats appeared obvious spatial learning and memory disorder; at the same time, the ovaries castrated group compared with normal group, the hippocampus glucose uptake was significantly decreased, the protein expression of GLUT4 in hippocampus decreased level of expression; mRNA and insulin decreased.2. protein in hippocampus of ovariectomized memory effect associated with estrogen receptor P receptor on spatial learning: Tamoxifen blocked ERp, Obviously impaired memory of rats spatial learning; at the same time, tamoxifen group hippocampus glucose uptake decreased expression of GLUT4 protein level in the hippocampus decreased, compared with the normal group; the tamoxifen group compared with normal group, the expression of mRNA and protein in hippocampus of ovariectomized.3. insulin decreased level of insulin and glucose on spatial learning and memory and hippocampus use the insulin antibody and hippocampus after insulin, compared with the normal group, the spatial learning and memory damage, the expression level of GLUT4 protein in hippocampus and hippocampal glucose uptake decreased significantly. Conclusion: estrogen on the hippocampus dependent learning and memory space is the role of estrogen receptor P (ER beta) complete: insulin signaling in hippocampus the process of estrogen effects on spatial learning and memory; in the process of spatial learning and memory in the hippocampus by estrogen insulin Levels reduce glucose metabolism in the hippocampus.

【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R711.75

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