雄激素对SAMP8小鼠学习记忆能力和海马神经元的影响

发布时间：2018-01-13 20:23

本文关键词：雄激素对SAMP8小鼠学习记忆能力和海马神经元的影响　出处：《河北医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的:应用SAMP8小鼠为研究对象,观察去势及雄激素补充治疗对其学习记忆能力和海马神经元的影响,初步探讨雄激素改善学习记忆能力的机制,为雄激素替代治疗提供实验依据。方法:选用健康雄性7月龄快速老化SAMP8小鼠48只。随机分为假手术对照组(简称P8组)、去势组、去势+雄激素补充治疗组(简称雄激素组)。选取16只雄性SAMR1小鼠作为同源正常对照组(简称R1组)。去势组和雄激素组小鼠切除睾丸。雄激素组于去势术1w后肌肉注射十一酸睾酮(TU)37.4mg?kg-1?15d-1,其余各组注射等量无菌芝麻油,注射3次,共45d。 1. Morris水迷宫试验:给药结束后,小鼠在迷宫实验室环境下饲养2d。训练在每天上午8:00～12:00间进行。定位航行试验:将Morris水迷宫均分为4个象限,平台放入其中1个象限中。小鼠自第一象限中点面向池壁入水,视频采集系统跟踪并记录找到平台所需的时间。5d后撤掉平台进行探索试验,记录小鼠120s内跨越平台的次数。 2.组织切片制备及观察:每组取10只小鼠,将输液针刺入左心室,同时剪开右心耳,用生理盐水冲洗,4%多聚甲醛灌注。自上丘至视交叉节段取脑组织,制备五套石蜡切片,分别用于HE染色,Nissl染色,TUNEL染色,抗Aβ免疫组化染色和阴性对照。 3.流式细胞仪检测海马细胞凋亡:每组取5只小鼠,断头取脑,迅速于冰盘上剥离海马组织,4℃70%乙醇中固定。采用网搓法制备单细胞悬液,碘化丙啶进行染色,流式细胞仪进行检测。 4.海马CA1区神经元超微结构观察:迅速游离海马,置于4%预冷的戊二醛溶液中固定,将海马切成厚约1mm的横断薄片,1h后再次切成1mm3的海马CA1区组织块,锇酸固定,脱水,树脂包埋,LKB-5超薄切片机切片,厚度50nm。醋酸铀-柠檬酸铅染色,透射电镜观察。结果: 1. Morris水迷宫实验结果:去势组定位航行试验潜伏期明显长于其他组(P0.05),探索试验跨越平台次数减少(P0.05)。TU补充治疗能改善学习记忆能力,与假手术组比较差异无统计学意义(P0.05)。 2. HE染色结果:R1组海马CA1区结构正常,神经元排列紧密整齐,层次清楚。P8组神经元排列较疏松紊乱,细胞层数减少。去势组海马CA1区神经元弥漫性空泡变性,细胞排列疏松、紊乱,细胞肿胀,核深染、固缩现象明显。雄激素组神经元病理改变较去势组有明显改善。 3. Nissl染色结果:R1组海马神经元胞浆中尼氏体丰富致密,细胞数较多,与其它各组相比差异有统计学意义(P0.01)。P8组海马神经元尼氏体较稀疏。去势组海马神经元数目较P8组明显减少,细胞核固缩、浓密深染,胞浆内尼氏体减少,部分缺失。雄激素补充治疗后,细胞数较去势组增加,差异有统计学意义(P0.05),但与P8组相比差异无统计学意义(P0.05)。 4. TUNEL染色结果:R1组存在少量散在的TUNEL阳性细胞。P8组阳性细胞数较R1组明显增加(P0.05)。去势组存在大量强阳性细胞,且核边界清晰,大小不一,多有核周空晕,TUNEL阳性细胞较P8组明显增加,差异有统计学意义(P0.05)。雄激素组阳性细胞表达较去势组减弱(P0.05),与P8组相比无统计学差异(P0.05)。 5.抗Aβ免疫组化染色结果:去势组Aβ阳性神经元染色深,其数目及吸光度明显高于其他组(P0.05)。雄激素组Aβ阳性细胞数目及吸光度较去势组明显减少(P0.05)。 6.流式细胞仪检测结果:去势组出现了一个较高的亚二倍体峰,细胞凋亡率达到32.66±4.60%,与P8组凋亡率21.19±3.96%以及雄激素组凋亡率20.78±3.08%相比明显升高,有统计学意义(P0.05),而P8组和雄激素组间凋亡率比较,差异无统计学意义(P0.05)。 7.电镜超微结构观察结果:电镜观察R1组神经元核膜完整光滑,核内染色质均匀,电子密度低,线粒体丰富。P8组神经元核膜双层结构较完整,线粒体正常或轻微肿胀,有少量脂褐素沉积,核内染色质电子密度略高。去势组神经元核膜双层结构消失融散,线粒体肿胀变性,嵴断裂,脂褐素沉积,核内染色质聚集成团、边集,形成不规则的高电子密度团块,有些出现凋亡小体。雄激素组超微结构损伤较去势组减轻,神经元核膜较清晰,核内染色质边集不明显,线粒体形态未见明显异常,脂褐素沉积减少。结论: 1. SAMP8小鼠去势后,雄激素缺乏导致学习记忆能力下降;雄激素补充治疗能减轻内源性雄激素下降对学习记忆功能的损害,改善学习记忆能力。 2. SAMP8小鼠去势后,雄激素缺乏导致海马神经元疏松紊乱、肿胀变性,超微结构受损,凋亡数目增加,神经元大量丢失;雄激素补充治疗能改善病理损伤,减轻神经元的凋亡和丢失。 3. SAMP8小鼠去势后,雄激素缺乏导致海马CA1区神经元Aβ沉着增加;雄激素补充治疗能减少Aβ的生成。
[Abstract]:Objective: To observe the effects of castration and androgen supplementation on learning and memory ability and hippocampal neurons in SAMP8 mice, and to preliminarily explore the mechanism of androgen improving learning and memory ability, so as to provide experimental evidence for androgen replacement therapy.
Methods: healthy male July age fast aging SAMP8 mice 48. Were randomly divided into sham operation group (P8 group), ovariectomized group, ovariectomized and androgen replacement therapy group (the androgen group). A total of 16 male SAMR1 mice were used as homologous normal control group (R1 group), ovariectomized group and androgen group mouse testis androgen group. Resection of castration 1W after intramuscular injection of eleven acid testosterone (TU) 37.4mg? Kg-1? 15d-1, other groups were injected with sterile sesame oil, 3 injection, 45d.
1. Morris water maze test: after injection, mice in the maze laboratory environment feeding 2D. training in the morning at 8:00 ~ 12:00. The place navigation test: the Morris water maze was divided into 4 quadrants, the platform into which of the 1 quadrants. Mice from the first quadrant midpoint to the wall into the water, video acquisition system to track and record the time required to find the platform after.5d removed platform experiment, record mouse 120s across platform number.
2. tissue sections were prepared and observed: each group of 10 mice, the infusion needle is inserted into the left ventricle, and cut the right atrial appendage, flushed with saline and 4% paraformaldehyde perfusion. From the superior colliculus to optic chiasma segment brain tissues were collected, prepared five sets of paraffin sections were used for HE staining, Nissl staining color, TUNEL staining, immunohistochemical staining of anti A beta and negative control.
Detection of apoptosis of the hippocampus cells. 3. flow cytometry: each group 5 mice were decapitated rapidly, in Bingpan stripping the hippocampus, fixed 4 C 70% ethanol. The rubbing preparation of single cell suspension, propidium iodide staining and flow cytometry.
To observe the ultrastructure of neurons in hippocampal CA1 area: 4. fast free hippocampus, fixed in 4% glutaraldehyde solution precooling, will cut into transverse slice thickness of 1mm hippocampus, 1H again after cut 1mm3 in CA1 area of hippocampus tissue, osmic acid fixation, dehydration, resin embedding, LKB-5 ultrathin sections, the thickness of 50nm. acetate uranium - lead citrate staining and transmission electron microscopy.
Result:
The results of 1. Morris water maze test showed that the latency of positioning test in ovariectomized group was significantly longer than that in other groups (P0.05), and the number of crossing platforms was reduced (P0.05)..TU supplementation treatment could improve learning and memory ability, and there was no significant difference between sham operation group and sham operation group (P0.05).
2. HE staining: group R1 normal neuronal structure in hippocampal CA1 area, arranged, clear.P8 group of neurons arranged in loose disorder, cell layers decreased. Castration groups of neurons in hippocampal CA1 region of diffuse vacuolar degeneration, cells arranged in loose and disorder, cell swelling, hyperchromatic nuclei pyknosis obviously. Androgen group of neurons the pathological change is significantly better than that of OVX group.
3. Nissl staining: R1 group of hippocampal neurons Nissl body rich and dense, cell number compared with the other groups there were statistically significant differences (P0.01).P8 group Nissl body in hippocampus were sparse. The number of neurons in the hippocampus of ovariectomized group decreased significantly compared with group P8, karyopyknosis, dense hyperchromatic cytoplasm tigroid the body is reduced, partial deletion. Androgen replacement therapy, cell number compared with OVX group increased, the difference was statistically significant (P0.05), but compared with the P8 group, the difference was not statistically significant (P0.05).
4. TUNEL staining: in group R1, TUNEL positive cells in.P8 group the number of positive cells in a few scattered was higher than R1 group (P0.05). A large number of strong positive cells in the ovariectomized group, and the nuclear size, clear boundary, a perinuclear halo, TUNEL positive cells were significantly increased compared with group P8, there were significant differences (P0.05). The expression of positive cells compared with androgen group (P0.05), ovariectomized group decreased compared with the P8 group showed no significant difference (P0.05).
5., anti A beta immunohistochemical staining results: the A beta positive neurons in the ovariectomized group were deep stained, the number and absorbance of them were significantly higher than those in other groups (P0.05). The number and absorbance of A beta positive cells in androgen group were significantly decreased compared with those in ovariectomized group (P0.05).
The testing results of 6. flow cytometry: the OVX group had a higher hypodiploid peak, the apoptosis rate of 32.66 + 4.60%, compared with the P8 group the apoptosis rate of 21.19 + 3.96% and 20.78 + 3.08% in the testosterone group apoptosis rate increased significantly, with statistical significance (P0.05), and P8 group and male hormone group apoptosis rate comparison, the difference was not statistically significant (P0.05).
7. ultrastructural observations: observation group R1 neurons nuclear membrane smooth and complete, the nuclear chromatin is uniform, low electron density, abundant mitochondria in neurons of the.P8 group double membrane structure is more complete, normal or slightly swollen mitochondria, a small amount of lipofuscin deposition, nuclear chromatin slightly higher electron density. The OVX group neurons double membrane structure disappearance of melting powder, mitochondria swelling, cristae, lipofuscin deposition, nuclear chromatin aggregation, margination, formation of high electron density were irregular, some apoptotic bodies were observed. The ultrastructure injury of androgen group than the OVX group decreased, nuclear membrane was clear, the nuclear chromatin is not obvious, mitochondria there was no obvious morphological abnormalities, lipofuscin deposition decreased.
Conclusion:
1., after SAMP8 castration, androgen deficiency leads to a decrease in learning and memory ability. Androgen supplementation therapy can reduce the impairment of endogenous androgen and damage learning and memory function, and improve learning and memory ability.
2. after SAMP8 castration, androgen deficiency resulted in loose, disorder, swelling, degeneration and ultrastructure of hippocampal neurons. The number of apoptotic neurons increased and the number of neurons was lost. Androgen supplementation therapy can improve pathological damage and alleviate neuronal apoptosis and loss.
After the castration of 3. SAMP8 mice, androgen deficiency leads to the increase of A beta in the hippocampal CA1 region neurons, and androgen supplement therapy can reduce the formation of A beta.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R341

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