雄激素对原代海马神经元树突棘及突触蛋白的快速作用

发布时间：2018-06-19 17:31

本文选题：雄激素 + 原代海马神经元　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:以原代培养的大鼠海马神经元为研究对象,观察睾酮(T)、双氢睾酮(DHT)和睾酮-牛血清白蛋白(T-BSA)对原代海马神经元树突棘的密度和形态以及突触蛋白PSD95和SYN的快速作用,探讨雄激素非基因组效应对海马神经元突触形态可塑性的影响。方法:1基于树突棘形态学观察的大鼠原代海马神经元转染GFP方法的优化。选用孕18 d的SD大鼠,解剖胎鼠脑组织,分离海马,进行海马神经元的培养。应用Lipofectamine 2000和慢病毒两种转染方法对体外培养8 d的SD大鼠海马神经元进行绿色荧光蛋白GFP质粒转染,观察海马神经元转染效率和荧光表达情况,并用台盼蓝染色检测神经元的存活率;之后,采用优选出的转染方法对体外培养8 d的海马神经元进行转染,观察转染后培养至10 d、15 d、20 d和25 d不同时间海马神经元树突棘的生长发育情况;最后,观察8 d、12 d和16 d不同时间转染对海马神经元树突棘形态的影响。2雄激素对大鼠原代海马神经元树突棘形态以及密度和表面积的快速影响。大鼠原代海马神经元培养。选取优化的转染方法Lipofectamine2000对海马神经元进行GFP转染,培养至第20 d时,利用共聚焦显微镜和活细胞工作站对转染成功且状态良好的海马神经元树突棘进行活细胞成像观察。实验随机分为4组:对照组(Con组)、T组、DHT组和T-BSA组。根据参考文献和预实验结果,T组和DHT组药物作用浓度为10 nM,T-BSA组为0.36 nM,Con组给予等量的二甲基亚砜。利用激光共聚焦显微镜观察药物干预前后树突棘的形态变化并记录树突棘的密度和表面积。每隔30 min扫描拍照一次,共观察120 min。3雄激素对大鼠原代海马神经元突触蛋白PSD95和SYN的快速影响。大鼠原代海马神经元培养。培养至第20 d时,进行实验。实验分组和药物作用浓度同上一部分。根据预实验结果,药物作用时间为60 min。给药结束后,应用免疫荧光细胞染色方法,激光共聚焦显微镜对突触蛋白psd95和syn进行观察。结果:1基于树突棘形态学观察的大鼠原代海马神经元转染gfp方法的优化。对于树突棘形态学观察而言,lipofectamine2000转染效果优于慢病毒,lipofectamine2000转染效率为5.21%,低于慢病毒转染效率82.53%。但就转染前后神经元存活率而言,lipofectamine2000转染后24h存活率为93.78%,高于慢病毒转染后24h存活率83.37%。转染1w后,lipofectamine2000转染存活率为91.59%,显著高于慢病毒转染存活率72.92%;且lipofectamine2000转染成功的神经元生长状态也优于慢病毒转染组。海马神经元培养至20d时,树突棘发育较成熟,丝状伪足明显减少,形成密集的短粗状或蘑菇状的树突棘。12d时进行转染是进行树突棘形态学观察的最佳转染时间;细胞培养至20d时荧光表达较好,树突棘形态清晰可见,适合进行活细胞树突棘长时间形态学观察。2雄激素对大鼠原代海马神经元树突棘形态以及密度和表面积的快速影响。树突棘形态:各组树突棘形态多样,蘑菇状、短粗状、细杆状等各种形状的树突棘可以在同一时间的同一树突上被发现,而且树突棘的形态不是固定不变的。在观察的时间内,树突棘的形态不是固定不变的。各组相比,con组的树突棘形态相对稳定,没有明显的变化。其它三组给药后,一些树突棘形态有了较明显的变化,更趋于成熟,表现为由细杆型变成短粗型或者由短粗型变成蘑菇型。树突棘密度:在120min的观察时间内,con组、t组、dht组和t-bsa组树突棘密度变化无统计学差异(p0.05)。树突棘表面积:con组树突棘的表面积随着时间的改变,相对稳定变化不大。t组、dht组和t-bsa组树突棘的表面积随着时间的改变,呈现一定的变化。在60min时,与con组(1.27±0.41)相比,t组(2.47±0.64)、dht组(2.68±1.08)和t-bsa组(2.61±0.67)树突棘表面积变化差异有统计学意义(p0.05)。在90min时,与con组(1.41±0.31)相比,t组(2.41±0.52)、dht组(2.58±0.80)和t-bsa组(2.38±0.60)树突棘表面积变化差异有统计学意义(p0.05)。其中,给药60min时,差异最显著。其余各时间段差异无统计学意义(P0.05)T组、DHT组和T-BSA组三组之间树突棘表面积变化无统计学差异(P0.05)。3雄激素对大鼠原代海马神经元突触蛋白PSD95和SYN的快速影响。PSD95免疫荧光细胞化学染色结果显示:与Con组(63.60±2.06)相比,T组(90.78±2.83)、DHT组(85.48±2.41)和T-BSA组(88.42±3.40)突触蛋白PSD95的荧光强度都有所增加,差异有统计学意义(P0.05)。T组、DHT组和T-BSA组三组之间PSD95荧光强度无统计学差异(P0.05)。SYN免疫荧光细胞化学染色结果显示:与Con组(52.96±0.97)相比,T组(63.02±0.66)、DHT组(65.00±1.46)和T-BSA组(63.47±1.05)突触蛋白SYN的荧光强度都有所增加,差异有统计学意义(P0.05)。T组、DHT组和T-BSA组三组之间SYN荧光强度无统计学差异(P0.05)。结论:1基于树突棘形态学观察的目的,用Lipofectamine 2000转染GFP方法对培养12 d的原代海马神经元进行转染后培养至20 d时进行树突棘形态学观察效果良好。2雄激素在短时间内未能增加大鼠原代海马神经元树突棘密度,但影响了树突棘形态,使树突棘更趋于成熟,并增加了树突棘表面积。3雄激素在短时间内快速增加了大鼠原代海马神经元突触蛋白PSD95和SYN的表达。
[Abstract]:Objective: To study the primary cultured rat hippocampal neurons, to observe the density and morphology of the dendritic spines in the primary hippocampal neurons and the rapid action of the synaptic protein PSD95 and SYN by testosterone (T), dihydrotestosterone (DHT) and testosterone bovine serum albumin (T-BSA), and to explore the synaptic plasticity of the androgen non genomic effect on the hippocampal neurons. Methods: 1 Optimization of the GFP method of rat primary hippocampal neurons based on the morphological observation of dendritic spines. The SD rats with 18 D pregnancy were selected to dissected the fetal rat brain tissue, the hippocampus was isolated and the hippocampal neurons were cultured. Lipofectamine 2000 and two lentivirus transfection methods were applied to the hippocampal neurons of SD rats cultured in vitro for 8 d in vitro. The transfection efficiency and fluorescence expression of hippocampal neurons were observed by the transfection of green fluorescent protein GFP plasmid, and the survival rate of neurons was detected by trypan blue staining. After the transfection, the cultured hippocampal neurons cultured in vitro were transfected with the preferred transfection method, and the hippocampal neurons were cultured to 10 d, 15 D, 20 D and 25 d at different time, and the hippocampal neurons were cultured at different time. The growth and development of spinous spines; finally, the effects of 8 D, 12 d and 16 D on the morphology of dendritic spines in hippocampal neurons were observed..2 androgen had a rapid effect on the morphology, density and surface area of the dendritic spines in the primary hippocampal neurons of rats. The primary cultured hippocampal neurons were cultured in rats. The optimized transfection method, Lipofectamine2000, was selected for the hippocampus. The neurons were transfected with GFP and cultured to twentieth D. A confocal microscope and a living cell workstation were used to observe the living cells of the dendritic spines of the hippocampal neurons, which were successfully transfected. The experiment was randomly divided into 4 groups: the control group (group Con), the T group, the DHT group and the T-BSA group. According to the reference and pre experimental results, the T group and DHT group drugs were made. The concentration of 10 nM, the T-BSA group was 0.36 nM, the Con group was given the equivalent two methyl sulfoxide. The morphological changes of dendritic spines before and after the drug intervention were observed by laser confocal microscopy and the density and surface area of the dendritic spines were recorded. A total of 30 min scans were taken each other, and the synaptic protein PSD95 of the primary hippocampal neurons of the rat was observed by 120 min.3 androxisin. And the rapid effect of SYN. Rat primary hippocampal neurons were cultured. When cultured to twentieth D, the experiment was carried out. The experimental group and the concentration of drug action were the same. According to the results of the pre experiment, after the drug action time was 60 min., the immunofluorescent cell staining method was used, the laser confocal microscope was performed on the synaptic protein PSD95 and syn. Results: 1 Optimization of the transfection of the primary hippocampal neurons based on the morphological observation of the dendritic spines. For the morphological observation of the dendritic spines, the transfection efficiency of lipofectamine2000 was better than that of the lentivirus, and the transfection efficiency of lipofectamine2000 was 5.21%, which was lower than that of the lentivirus transfection efficiency 82.53%., but the survival rate of the neurons before and after the transfection was Li. Li After transfection of pofectamine2000, the survival rate of 24h was 93.78%. The survival rate of lipofectamine2000 transfected after transfection of lentivirus was higher than that of 1W, the survival rate of lipofectamine2000 transfection was 91.59%, which was significantly higher than the survival rate of lentivirus transfection by 72.92%, and the successful neuronal growth of lipofectamine2000 transfection was also better than that of the lentivirus transfection group. The hippocampal neurons were cultured to 20d. When the dendritic spines developed more mature, the filamentous pseudo foot decreased obviously, and the dense short or mushroom like dendritic spines were formed when the transfection was the best transfection time for the morphological observation of the dendritic spines. The cell culture to 20d showed good fluorescence expression and the morphology of the dendritic spines was clearly visible. It was suitable for the long time morphological observation of the dendritic spines. 2 the rapid effects of androgen on the dendritic spines, density and surface area of the primary hippocampal neurons in the rat. The morphology of the dendrite spines: the various dendritic spines in each group were found in the same dendrite at the same time, and the dendritic spines were not fixed. During the time, the morphology of the dendrite spines was not fixed. Compared with each group, the dendritic spines in the con group were relatively stable, and there was no obvious change. After the other three groups, some dendritic spines had a more obvious change, more mature, and the dendrite density was 1, and the dendrite density: the density of the dendrite spines was 1. During the observation time of 20min, there was no significant difference in dendrite density between group con, t, DHT and t-bsa. The surface area of dendrite spines in group con: the surface area of dendritic spines in con group changed little with time, and the surface area of dendritic spines in DHT and t-bsa groups changed with time. Group (1.27 + 0.41), group t (2.47 + 0.64), DHT group (2.68 + 1.08) and t-bsa group (2.61 + 0.67) have significant difference in dendrite surface area (P0.05). At 90min, compared with group con (1.41 + 0.31), t group (2.41 + 0.52), DHT group (2.58 + 0.80) and t-bsa group (P0.05), there were significant differences in dendrite surface area (P0.05). The difference was most significant when 60min was given. There was no significant difference between the other time periods (P0.05) T group, and there was no statistical difference between the three groups of DHT and T-BSA groups (P0.05) the rapid effect of.3 androgen on the synaptic protein PSD95 and SYN in the primary hippocampal neurons of the rat.PSD95 immunofluorescence staining results of.PSD95: and Con Group (63.60 + 2.06), group T (90.78 + 2.83), DHT group (85.48 + 2.41) and T-BSA group (88.42 + 3.40), the fluorescence intensity of PSD95 increased, the difference was statistically significant (P0.05).T group. The PSD95 fluorescence intensity of DHT group and T-BSA group three groups was not statistically significant (P0.05).SYN immunofluorescent cytochemical staining results showed: and Con group (52 .96 + 0.97) compared with group T (63.02 + 0.66), DHT group (65 + 1.46) and T-BSA group (63.47 + 1.05) synaptic protein SYN fluorescence intensity increased, the difference was statistically significant (P0.05).T group, DHT and T-BSA group three groups of SYN fluorescence intensity no statistical difference (P0.05). Conclusion: 1 based on the morphological observation of dendritic spines, with Lipofectamine 200 0 transfection of GFP method to culture 12 d primary hippocampal neurons after transfection and culture to 20 D, the morphological observation of dendritic spines was good,.2 androgen did not increase the dendrite density of the primary hippocampal neurons in a short time, but it affected the morphology of the dendritic spines, made the dendrite spines more mature, and increased the surface area of the dendritic spines,.3 male. Hormone increased the expression of synaptophysin PSD95 and SYN in primary hippocampal neurons in a short time.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R338

【参考文献】