穹窿切断后大鼠海马神经元MR、GR表达变化的研究

发布时间：2018-05-23 16:55

本文选题：穹窿切断术 + MR　；参考：《中国医科大学》2007年硕士论文

【摘要】： 目的下丘脑—垂体—肾上腺(hypothalamic-pituitary-adrenal，HPA)轴作为神经内分泌免疫网络的枢纽，在机体内环境平衡维持及机体衰老过程的调节中起着十分重要的作用。糖皮质激素作用于垂体和下丘脑构成HPA轴负反馈调节的主要通路，然而现有大量资料表明，下丘脑以上结构特别是海马参与了HPA轴的调节，而且是具有重要意义的负反馈调节。目前的研究普遍认为海马通过赫皮质激素受体(Mineralocorticoid Receptor，MR)和糖皮质激素受体(Glucocorticoid Receptor，GR)对下丘脑的室旁核产生了抑制作用。静息条件下，糖皮质激素与海马中的MR结合参与基础水平的HPA轴负反馈调节；应激条件下，高水平的应激激素与海马中的GR结合，抑制HPA轴的过度反应或使应激状态的HPA轴功能恢复到基础水平。MR、GR这两个受体属于核受体家族，核受体是能与特异性激素结合的蛋白，主要是一类配基依赖性转录调节因子，通过基因表达诱导或者抑制细胞的增殖，分化和死忙，导致多种生物效应。但是从海马到室旁核没有直接的神经投射，间接的路径都要经过穹窿。现有研究报道穹窿切断也可以影响室旁核的CRH和AVP的水平，由此引发我们推测海马对下丘脑室旁核的抑制作用是否媒介了穹窿。本实验建立大鼠穹窿切断模型，采用免疫组织化学、western blotting、图像分析等方法对穹窿切断后大鼠海马神经元MR、GR的表达进行了研究，希望为海马神经元MR、GR调节HPA轴的相关机制提供实验依据。实验方法一、实验动物及分组成年健康雄性Wistar大鼠90只，体重200—220g，中国医科大学实验动物中心提供，，实验室条件饲养。随机分为正常对照组(10只)、穹窿切断组(分为0天、4天、7天、10天组，每组10只)、假手术组(未切穹窿，分为0天、4天、7天、10天组，每组10只) 二、动物模型的建立将大鼠用戊巴比妥将其麻醉后，固定于脑立体定位仪(江湾IC型)，常规消毒、剪毛，正中切开头皮，暴露出颅骨，于前囟后1.5mm，中线外左右1.0mm处，用电动开颅器凿开颅骨，切开硬脑膜，用自制的双刃刀先置于上述部位的脑表面，(相当于穹窿部位)，续降刀6mm，切断穹窿，降刀时间约1分钟，假手术组降刀2mm，(未切穹窿)，刀在脑中均停留5分钟。清洁颅面，缝合头皮，常规饲养，抗菌素控制感染。三、大鼠穹窿切断模型的鉴定对正常组和穹窿切断组大鼠进行海马胆碱能纤维AchE组织化学染色。四、免疫组织化学染色各组大鼠经4％多聚甲醛灌流固定、取材，制作冰冻切片进行免疫组化SABC法染色，(MR工作浓度为1：200、GR工作浓度为1：500)，DAB显色，中性树胶封片，光学显微镜(OLYMPUS，BX60，Japan)下观察，摄片。并作图像分析。五、Western Blotting 取新鲜海马组织经匀浆、超声粉碎后高速低温离心，提取上清蛋白，电泳，转膜，经封闭后加一抗MR、GR(MR工作浓度1：500，GR工作浓度1：1000)4℃过夜；HRP标记IgG(工作浓度1：1500)孵育2h，DAB法显色，扫描条带计算灰度值。实验结果一、大鼠海马纤维AchE染色结果正常海马结构内有丰富的胆碱能纤维，纤维粗细均匀且交织成网。穹窿切断组AchE阳性纤维大量丧失，观察海马CA1区神经纤维，纤维变细排列紊乱且密度降低。说明穹窿切断模型建立成功。二、NR、GR免疫组化反应染色结果正常组MR、GR免疫反应阳性产物呈棕黄色表达，位于细胞质和细胞核内，MR主要位于海马CA1、CA2、CA3区的锥体细胞层和齿状回的颗粒细胞层的神经元。GR主要位于海马CA1、CA2区的锥体细胞层和齿状回的颗粒细胞层的神经元。观察海马CA1区，MR、GR表达阳性的锥体细胞数目较多，染色较深，细胞排列较整齐。各假手术组MR、GR的表达与正常组相似，0、4天穹窿切断组与正常组、相应假手术组相比MR、GR免疫反应阳性细胞的数量与形态没有明显改变，7、10天穹窿切断组海马CA1区MR、GR免疫反应阳性细胞数较少，细胞形态变小，染色浅。而且10天穹窿切断组较7天穹窿切断组免疫反应阳性细胞数少，染色更浅。三、MR、GR Western BIotting结果 Western Blotting检测发现海马MR、GR的表达在7天、10天穹窿切断组较正常组、假手术组减少，且10天组较7天组表达量少。0、4天组与正常组、假手术组相比无明显差异。各假手术组MR、GR的表达与正常组相比无明显差异。四、图像分析结果 7天、10天穹窿切断组，海马神经元MR、GR表达较正常组、假手术组表达量减少(p＜0.05)，10天较7天组表达量减少(p＜0.05)0、4天穹窿切断组与正常组、假手术组相比无明显差异(p＞0.05)。各假手术组MR、GR的表达与正常组相比无明显差异(p＞0.05)。结论穹窿切断后，大鼠海马神经元MR、GR表达减弱，提示可能穹窿切断后海马对HPA轴的抑制作用减弱。
[Abstract]:objective
The hypothalamus pituitary adrenal (hypothalamic-pituitary-adrenal, HPA) axis, as the hub of the neuroendocrine immune network, plays a very important role in the maintenance of environmental balance and the regulation of the aging process in the body. Glucocorticoid acts as the main pathway of HPA axis negative feedback regulation in the pituitary and hypothalamus, but now it is the main pathway of the negative feedback regulation of the pituitary and hypothalamus. A large number of data indicate that the hypothalamic structure, especially the hippocampus, is involved in the regulation of the HPA axis, and is a significant negative feedback regulation.
At present, it is widely believed that the hippocampus inhibits the paraventricular nucleus of the hypothalamus through the Mineralocorticoid Receptor (MR) and Glucocorticoid Receptor (GR). In resting conditions, the glucocorticoid and the MR in the hippocampus are involved in the negative feedback regulation of the HPA axis of the basal level; the stress bar is a stress bar. A high level of stress hormone combined with GR in the hippocampus to inhibit the overreaction of the HPA axis or to restore the HPA axis function of the stress state to the basic level.MR. The two receptors of GR belong to the nuclear receptor family, and the nuclear receptor is a protein binding to the specific hormone, mainly a class of ligand dependent transcription factors, which are induced by gene expression. The proliferation, differentiation, and death of cells lead to a variety of biological effects. But there is no direct neural projection from the hippocampus to the paraventricular nucleus and the indirect path passes through the fornix. The present study reports that the fornix cut can also affect the level of CRH and AVP in the paraventricular nucleus, which leads us to speculate on the inhibition of the hippocampus to the paraventricular nucleus of the inferior colliculus. Whether the action mediate the fornix.
In this experiment, rat fornix cutting model was established. Immunohistochemistry, Western blotting and image analysis were used to study the expression of MR and GR in hippocampal neurons of rats after fornix fornix cutting. It was hoped to provide experimental evidence for the mechanism of MR and GR to regulate the HPA axis of hippocampal neurons.
Experimental method
First, experimental animals and groups
90 adult healthy male Wistar rats, weighing 200 - 220g, were provided by laboratory animal center of China Medical University and kept in laboratory. They were randomly divided into normal control group (10 rats), fornix cutting group (divided into 0 days, 4 days, 7 days, 10 days, 10 in each group), and the sham operation group (0 days, 4 days, 7 days, 10 days, 10 only)
Two, the establishment of animal model
The rats were anaesthetized with pentobarbital, fixed to the brain stereotaxis (Jiangwan type IC), routinely sterilizing, cutting hair, cutting the scalp in the midline, exposing the skull, 1.5mm in the anterior fontanel, and outside the middle line 1.0mm. The skull was cut open and the dura was cut with an electric craniator, and the surface of the brain was first placed with a self-made double-edged knife (equivalent to the fornix). 6mm, cut the fornix, cut the fornix, cut the knife time about 1 minutes, the sham operation group reduced the knife 2mm, (not cut the fornix), the knife stayed in the brain for 5 minutes. Clean the craniofacial, suture the scalp, the routine feeding, antibiotics control infection.
Three, identification of fornix fornix model in rats
The hippocampal cholinergic fibers of rats in the normal group and fornix transection group were stained with AchE histochemistry.
Four, immunohistochemical staining
The rats were perfused and fixed by 4% polyformaldehyde in each group. The frozen sections were made by immunohistochemical SABC staining. (MR working concentration was 1:200, GR working concentration was 1:500), DAB color, neutral gum seal, optical microscopy (OLYMPUS, BX60, Japan) were observed and photographed, and image analysis was made.
Five, Western Blotting
The fresh hippocampal tissues were homogenized and centrifuged at high speed and low temperature after ultrasonic comminution. The supernatant protein, electrophoresis, and membrane were extracted. After closed, a anti MR, GR (MR working concentration 1:500, GR working concentration 1:1000) were 4 degrees centigrade; HRP labeled IgG (working concentration 1:1500) was incubated for 2H, DAB method was coloured, and the scanning strip was used to calculate gray value.
experimental result
The results of AchE staining in the hippocampus of rats
There were plenty of cholinergic fibers in the normal hippocampal structure. The fibrous thickness was uniform and interwoven into the net. The AchE positive fibers in the fornix fornix group were lost and the nerve fibers in the CA1 region of the hippocampus were observed. The fibers were arranged in a thin arrangement and the density was reduced.
Two, NR, GR immunohistochemical staining results
In the normal group MR, the positive products of the GR immunoreaction were brown and yellow, located in the cytoplasm and nucleus, and MR was mainly located in the hippocampus CA1. The pyramidal layer of the CA2, CA3 region and the granular cell layer of the dentate gyrus were mainly located in the hippocampal CA1, the pyramidal layer of the CA2 region and the granular cell layer of the dentate gyrus. The CA1 region of the hippocampus, MR, GR was observed. The number of positive pyramidal cells was more, the coloring was deeper and the cells were arranged neatly. The expression of MR and GR in the sham operation group was similar to that of the normal group. The number and the morphology of the 0,4 vault cut group were compared with the normal group and the corresponding sham operation group, compared with the corresponding sham operation group, the number and shape of the GR immunoreactive cells were not changed obviously. The CA1 region MR of the hippocampus of 7,10 days fornix fornix group and the GR immune response were found. The number of positive cells was smaller, cell morphology was smaller and staining was shallow. Moreover, the number of immunoreactive cells in the fornix cut group was less than that in the 7 day fornix transection group, and the staining was more shallow on the 10 day.
Three, MR, GR Western BIotting results
Western Blotting detected the expression of MR in the hippocampus, the expression of GR in 7 days, the 10 day of the fornix cutting group was less than the normal group, the sham operation group decreased, and the 10 day group compared with the 7 day group was less.0,4 days than the normal group, and there was no significant difference between the sham operation group and the sham operation group. The expression of GR in the sham operation group was not significantly different from that of the normal group.
Four, image analysis results
On the 7 day, the 10 day fornix cutting group, the hippocampal neurons MR, GR expression was more than the normal group, the sham operation group decreased (P < 0.05), the 10 day group was less than the 7 day group (P < 0.05) 0,4 dome cut group and the normal group, the sham operation group had no significant difference (P > 0.05). The expression of MR in the artificial hand group was not significantly different from that of the normal group (P > 0.05).
conclusion
After fornix transection, the expression of MR and GR in hippocampal neurons of rats was weakened, suggesting that the inhibitory effect of hippocampus on HPA axis might be weakened after fornix transection.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R322.811

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