發(fā)布時(shí)間：2018-06-19 17:31

  本文選題：雄激素 + 原代海馬神經(jīng)元��； 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:以原代培養(yǎng)的大鼠海馬神經(jīng)元為研究對(duì)象,觀察睪酮(T)、雙氫睪酮(DHT)和睪酮-牛血清白蛋白(T-BSA)對(duì)原代海馬神經(jīng)元樹突棘的密度和形態(tài)以及突觸蛋白PSD95和SYN的快速作用,探討雄激素非基因組效應(yīng)對(duì)海馬神經(jīng)元突觸形態(tài)可塑性的影響。方法:1基于樹突棘形態(tài)學(xué)觀察的大鼠原代海馬神經(jīng)元轉(zhuǎn)染GFP方法的優(yōu)化。選用孕18 d的SD大鼠,解剖胎鼠腦組織,分離海馬,進(jìn)行海馬神經(jīng)元的培養(yǎng)。應(yīng)用Lipofectamine 2000和慢病毒兩種轉(zhuǎn)染方法對(duì)體外培養(yǎng)8 d的SD大鼠海馬神經(jīng)元進(jìn)行綠色熒光蛋白GFP質(zhì)粒轉(zhuǎn)染,觀察海馬神經(jīng)元轉(zhuǎn)染效率和熒光表達(dá)情況,并用臺(tái)盼藍(lán)染色檢測(cè)神經(jīng)元的存活率;之后,采用優(yōu)選出的轉(zhuǎn)染方法對(duì)體外培養(yǎng)8 d的海馬神經(jīng)元進(jìn)行轉(zhuǎn)染,觀察轉(zhuǎn)染后培養(yǎng)至10 d、15 d、20 d和25 d不同時(shí)間海馬神經(jīng)元樹突棘的生長(zhǎng)發(fā)育情況;最后,觀察8 d、12 d和16 d不同時(shí)間轉(zhuǎn)染對(duì)海馬神經(jīng)元樹突棘形態(tài)的影響。2雄激素對(duì)大鼠原代海馬神經(jīng)元樹突棘形態(tài)以及密度和表面積的快速影響。大鼠原代海馬神經(jīng)元培養(yǎng)。選取優(yōu)化的轉(zhuǎn)染方法Lipofectamine2000對(duì)海馬神經(jīng)元進(jìn)行GFP轉(zhuǎn)染,培養(yǎng)至第20 d時(shí),利用共聚焦顯微鏡和活細(xì)胞工作站對(duì)轉(zhuǎn)染成功且狀態(tài)良好的海馬神經(jīng)元樹突棘進(jìn)行活細(xì)胞成像觀察。實(shí)驗(yàn)隨機(jī)分為4組:對(duì)照組(Con組)、T組、DHT組和T-BSA組。根據(jù)參考文獻(xiàn)和預(yù)實(shí)驗(yàn)結(jié)果,T組和DHT組藥物作用濃度為10 nM,T-BSA組為0.36 nM,Con組給予等量的二甲基亞砜。利用激光共聚焦顯微鏡觀察藥物干預(yù)前后樹突棘的形態(tài)變化并記錄樹突棘的密度和表面積。每隔30 min掃描拍照一次,共觀察120 min。3雄激素對(duì)大鼠原代海馬神經(jīng)元突觸蛋白PSD95和SYN的快速影響。大鼠原代海馬神經(jīng)元培養(yǎng)。培養(yǎng)至第20 d時(shí),進(jìn)行實(shí)驗(yàn)。實(shí)驗(yàn)分組和藥物作用濃度同上一部分。根據(jù)預(yù)實(shí)驗(yàn)結(jié)果,藥物作用時(shí)間為60 min。給藥結(jié)束后,應(yīng)用免疫熒光細(xì)胞染色方法,激光共聚焦顯微鏡對(duì)突觸蛋白psd95和syn進(jìn)行觀察。結(jié)果:1基于樹突棘形態(tài)學(xué)觀察的大鼠原代海馬神經(jīng)元轉(zhuǎn)染gfp方法的優(yōu)化。對(duì)于樹突棘形態(tài)學(xué)觀察而言,lipofectamine2000轉(zhuǎn)染效果優(yōu)于慢病毒,lipofectamine2000轉(zhuǎn)染效率為5.21%,低于慢病毒轉(zhuǎn)染效率82.53%。但就轉(zhuǎn)染前后神經(jīng)元存活率而言,lipofectamine2000轉(zhuǎn)染后24h存活率為93.78%,高于慢病毒轉(zhuǎn)染后24h存活率83.37%。轉(zhuǎn)染1w后,lipofectamine2000轉(zhuǎn)染存活率為91.59%,顯著高于慢病毒轉(zhuǎn)染存活率72.92%;且lipofectamine2000轉(zhuǎn)染成功的神經(jīng)元生長(zhǎng)狀態(tài)也優(yōu)于慢病毒轉(zhuǎn)染組。海馬神經(jīng)元培養(yǎng)至20d時(shí),樹突棘發(fā)育較成熟,絲狀偽足明顯減少,形成密集的短粗狀或蘑菇狀的樹突棘。12d時(shí)進(jìn)行轉(zhuǎn)染是進(jìn)行樹突棘形態(tài)學(xué)觀察的最佳轉(zhuǎn)染時(shí)間;細(xì)胞培養(yǎng)至20d時(shí)熒光表達(dá)較好,樹突棘形態(tài)清晰可見,適合進(jìn)行活細(xì)胞樹突棘長(zhǎng)時(shí)間形態(tài)學(xué)觀察。2雄激素對(duì)大鼠原代海馬神經(jīng)元樹突棘形態(tài)以及密度和表面積的快速影響。樹突棘形態(tài):各組樹突棘形態(tài)多樣,蘑菇狀、短粗狀、細(xì)桿狀等各種形狀的樹突棘可以在同一時(shí)間的同一樹突上被發(fā)現(xiàn),而且樹突棘的形態(tài)不是固定不變的。在觀察的時(shí)間內(nèi),樹突棘的形態(tài)不是固定不變的。各組相比,con組的樹突棘形態(tài)相對(duì)穩(wěn)定,沒有明顯的變化。其它三組給藥后,一些樹突棘形態(tài)有了較明顯的變化,更趨于成熟,表現(xiàn)為由細(xì)桿型變成短粗型或者由短粗型變成蘑菇型。樹突棘密度:在120min的觀察時(shí)間內(nèi),con組、t組、dht組和t-bsa組樹突棘密度變化無統(tǒng)計(jì)學(xué)差異(p0.05)。樹突棘表面積:con組樹突棘的表面積隨著時(shí)間的改變,相對(duì)穩(wěn)定變化不大。t組、dht組和t-bsa組樹突棘的表面積隨著時(shí)間的改變,呈現(xiàn)一定的變化。在60min時(shí),與con組(1.27±0.41)相比,t組(2.47±0.64)、dht組(2.68±1.08)和t-bsa組(2.61±0.67)樹突棘表面積變化差異有統(tǒng)計(jì)學(xué)意義(p0.05)。在90min時(shí),與con組(1.41±0.31)相比,t組(2.41±0.52)、dht組(2.58±0.80)和t-bsa組(2.38±0.60)樹突棘表面積變化差異有統(tǒng)計(jì)學(xué)意義(p0.05)。其中,給藥60min時(shí),差異最顯著。其余各時(shí)間段差異無統(tǒng)計(jì)學(xué)意義(P0.05)T組、DHT組和T-BSA組三組之間樹突棘表面積變化無統(tǒng)計(jì)學(xué)差異(P0.05)。3雄激素對(duì)大鼠原代海馬神經(jīng)元突觸蛋白PSD95和SYN的快速影響。PSD95免疫熒光細(xì)胞化學(xué)染色結(jié)果顯示:與Con組(63.60±2.06)相比,T組(90.78±2.83)、DHT組(85.48±2.41)和T-BSA組(88.42±3.40)突觸蛋白PSD95的熒光強(qiáng)度都有所增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。T組、DHT組和T-BSA組三組之間PSD95熒光強(qiáng)度無統(tǒng)計(jì)學(xué)差異(P0.05)。SYN免疫熒光細(xì)胞化學(xué)染色結(jié)果顯示:與Con組(52.96±0.97)相比,T組(63.02±0.66)、DHT組(65.00±1.46)和T-BSA組(63.47±1.05)突觸蛋白SYN的熒光強(qiáng)度都有所增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。T組、DHT組和T-BSA組三組之間SYN熒光強(qiáng)度無統(tǒng)計(jì)學(xué)差異(P0.05)。結(jié)論:1基于樹突棘形態(tài)學(xué)觀察的目的,用Lipofectamine 2000轉(zhuǎn)染GFP方法對(duì)培養(yǎng)12 d的原代海馬神經(jīng)元進(jìn)行轉(zhuǎn)染后培養(yǎng)至20 d時(shí)進(jìn)行樹突棘形態(tài)學(xué)觀察效果良好。2雄激素在短時(shí)間內(nèi)未能增加大鼠原代海馬神經(jīng)元樹突棘密度,但影響了樹突棘形態(tài),使樹突棘更趨于成熟,并增加了樹突棘表面積。3雄激素在短時(shí)間內(nèi)快速增加了大鼠原代海馬神經(jīng)元突觸蛋白PSD95和SYN的表達(dá)。
[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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R338

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 陳燕;;神經(jīng)元的突觸可塑性與學(xué)習(xí)和記憶[J];生物化學(xué)與生物物理進(jìn)展;2008年06期

2 蒲昭霞;趙聰敏;李亞伶;張雪瓊;;腦發(fā)育不同階段豐富環(huán)境刺激對(duì)大鼠海馬突觸素表達(dá)的影響[J];中國兒童保健雜志;2007年06期

3 韓太真;突觸可塑性與長(zhǎng)時(shí)程增強(qiáng)現(xiàn)象的研究進(jìn)展[J];西安交通大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2005年04期

，

本文編號(hào)：2040704