幼年和成年单眼形觉剥夺性弱视大鼠视皮质PSD-95表达的研究

发布时间：2018-06-03 20:56

本文选题：PSD-95 + 单眼形觉剥夺　；参考：《新乡医学院》2012年硕士论文

【摘要】：研究背景弱视是儿童时期常见的严重影响视功能发育的眼病,患病率约为1%-5%。弱视的发病机制与视觉神经元突触的发育和可塑性变化密切相关,突触可塑性的变化是形成弱视的中心环节。突触后致密蛋白-95(PSD-95)在调节突触发育和可塑性中起关键作用,在蛋白水平已证实PSD-95参与视觉早期的发育,而在基因水平的相关研究则相对较少。目的观察幼年和成年单眼形觉剥夺性弱视大鼠和正常发育大鼠视皮质中PSD-95的表达情况,从分子水平来探索弱视的发病机制,为临床预防与治疗弱视提供新的思路。方法 1选取14日龄SD大鼠48只,雌雄不限,随机分为单眼剥夺组(MD)24只和正常对照组(NC)24只。NC组随机分为正常组Ⅰ组(NC Ⅰ)、正常组Ⅱ组(NCⅡ),每组各12只；MD组随机分为剥夺组Ⅰ组(MD Ⅰ)、剥夺组Ⅱ组(MD Ⅱ),每组各12只。MD组所有大鼠在生后14天行单侧眼睑缝合术建立单眼剥夺模型。 2NC Ⅰ组和MD Ⅰ组大鼠共同饲养至生后45天(P45,幼年期)取材,所得标本为NCP45、MDP45;NCⅡ组和MDⅡ组大鼠均饲养至生后90天(P90,成年期)取材,所得标本为NCP90、MDP90, MD组取材前均经图形视觉诱发电位(P-VEP)检测证实造模成功。 3应用Nissl染色法观察各组大鼠视皮质神经元的形态改变并定位分层,采用免疫组织化学方法和逆转录聚合酶链反应技术(RT-PCR)检测各组大鼠视皮质中PSD-95蛋白表达和PSD-95mRNA转录水平的变化。 4通过摄像显微镜照相,图像分析系统进行图像分析得出PSD-95阳性反应产物的平均光密度(AOD)值和PSD-95mRNA的相对表达量,数据用SPSS12.0统计软件进行处理,以均数士标准差(x±s)表示,相同发育阶段的组间比较采用成组设计的t检验,不同发育阶段的组间比较采用方差分析。结果 1P-VEP检测结果：经P-VEP检测后可见,正常对照组大鼠P-VEP均有稳定的波形、潜伏期和振幅。在幼年期(P45)和成年期(P90),MD组剥夺眼与NC组大鼠眼、MD组未剥夺眼比较,其P-VEP的P波潜伏期延长、波形不稳定、波幅下降,差异均有显著性意义(P0.05)；在幼年期和成年期,MD组未剥夺眼与NC组大鼠眼比较,差异均无统计学意义(P0.05)。 2Nissl染色结果：NC组大鼠视皮质神经元呈空泡状,胞浆着色,核不着色。视皮质的神经元分布呈层状,从皮质浅层至深层依次为：分子层(Ⅰ)、外颗粒层(Ⅱ)、外锥体层(Ⅲ)、内颗粒层(Ⅳ)、内锥体层(Ⅴ)、多形细胞层(Ⅵ)。与NC组大鼠比较,MD组大鼠剥夺眼对侧视皮质Nissl染色结果未见明显异常。 3免疫组织化学染色结果：光镜下可见,PSD-95阳性反应产物主要表达在神经元的胞体中,呈棕色颗粒状广泛分布于视皮质各层。PSD-95阳性反应产物在NC组大鼠视皮质中表达密集,其AOD值在NCP45组为0.397±0.008,在NCP90组为0.318±0.007,两组间的差异有统计学意义(P0.05)；MD组大鼠视皮质中PSD-95阳性表达产物的数量均减少,密度均减低,其AOD值在MDP45组为0.352±0.005(P4550.01),在MDP90组为0.259±0.011(P900.01)；MD组各发育阶段间PSD-95阳性反应产物的表达差异有显著性(P0.05)。 4RT-PCR法检测结果：PSD-95mRNA在各组大鼠的视皮质中均有表达。PSD-95mRNA的相对表达量在NCP45组为0.667±0.009,在NCP90组为0.399±0.019,两组间的差异有显著性(P0.05)；不同发育阶段MD组与NC组比较,PSD-95mRNA的表达均有明显减少,其相对表达量在MDP45组为0.470±0.005(P450.01),在MDP90组为0.350±0.005(P900.01)；MD组各发育阶段间PSD-95mRNA的相对表达量差异有显著性(P0.05)。结论 1在视觉发育的关键期内,视觉经验通过调节视觉神经元间的突触联系来调控PSD-95的表达,PSD-95是弱视发病机制的重要分子生物学基础之一； 2异常视觉经验能够影响成年大鼠视皮质内PSD-95的表达,推测成年大鼠的视皮质神经元仍具有突触可塑性； 3与幼年期正常发育大鼠比较,成年期正常发育大鼠视皮质中PSD-95的表达减少,推测年龄因素是PSD-95表达的影响因素之一。
[Abstract]:Research background
Amblyopia is a common ocular disease affecting the development of visual function, which is about 1%-5%. amblyopia. The incidence of amblyopia is closely related to the development and plasticity of synapses in visual neurons. The change of synaptic plasticity is the central link in the formation of amblyopia. The postsynaptic dense protein -95 (PSD-95) plays a role in regulating synaptic development and plasticity. The key role of protein at the protein level has been confirmed that PSD-95 is involved in the early development of vision, while the relative research at the gene level is relatively small.
objective
To observe the expression of PSD-95 in the visual cortex of young and adult monocular deprived amblyopia and normal developing rats, and to explore the pathogenesis of amblyopia from the molecular level, and provide a new idea for the clinical prevention and treatment of amblyopia.
Method
1 14 day old SD rats were randomly divided into single eye deprivation group (MD) and normal control group (NC), and 24.NC groups were randomly divided into normal group I (NC I), normal group II Group (NC II) and each group 12. The MD group was randomly divided into group I (MD I) and group II (MD II) in deprivation group (MD II), and all rats in each group of 12.MD groups were 14 days after birth. Unilateral eyelid suture was performed to establish a monocular deprivation model.
Group 2NC I and group MD I were reared together to 45 days after birth (P45, young). The specimens were NCP45, MDP45, NC II and MD II rats were reared to 90 days after birth (P90, adulthood). The specimens were tested by the visual evoked potential (P-VEP) test before the samples were obtained from NCP90, MDP90, MD group.
3 the morphological changes of the cortical neurons of the rats were observed by Nissl staining and the stratification was located. The changes of the expression of PSD-95 protein and the level of PSD-95mRNA transcription in the visual cortex of each group were detected by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR).
4 the average light density (AOD) value of PSD-95 positive reaction products and the relative expression of PSD-95mRNA were obtained by image analysis system by image analysis system. The data were processed with SPSS12.0 statistical software, with the standard deviation of the number of men (x + s), and the group designed t test was used in the same development stage, and the difference between the groups was different. Variance analysis is used in the comparison between groups at the developmental stage.
Result
1P-VEP detection results: after P-VEP detection, the normal control group showed that P-VEP had stable waveform, latent period and amplitude. In juvenile (P45) and adult stage (P90), MD group deprived eye and NC group eyes, and MD group was not deprived of eye, the latency of P-VEP P wave extended, wave instability, amplitude decreased, the difference had significant significance (P0.05). In the juvenile and adulthood, there was no significant difference between the MD group and the NC group (P0.05).
2Nissl staining results: the neurons of the visual cortex in group NC were vacuolated, cytoplasm coloring and non coloring. The distribution of neurons in the visual cortex was stratified, from the superficial layer to the deep layer: the molecular layer (I), the outer layer (II), the outer pyramidal layer (III), the inner granular layer (IV), the inner pyramidal layer (V) and the multiform cell layer (VI). Compared with the NC group, the MD group was larger. There was no obvious abnormality in Nissl staining of lateral eye cortex in rat deprived eyes.
3 immunohistochemical staining results: the results showed that the PSD-95 positive reaction products were mainly expressed in the cell body of the neurons, and the.PSD-95 positive reaction products were widely distributed in the visual cortex of the NC group, and the AOD value was 0.397 + 0.008 in the NCP45 group and 0.318 + 0.007 in the NCP90 group and in the two groups. The difference was statistically significant (P0.05). The number of PSD-95 positive products in the visual cortex of group MD decreased and the density decreased. The AOD value was 0.352 + 0.005 (P4550.01) in the MDP45 group and 0.259 + 0.011 (P900.01) in the MDP90 group, and the expression difference between the PSD-95 positive reaction products of the MD group was significant (P0.05).
The results of 4RT-PCR assay: the relative expression of.PSD-95mRNA in the visual cortex of the rats of each group was 0.667 + 0.009 in the NCP45 group and 0.399 + 0.019 in the NCP90 group. The difference between the two groups was significant (P0.05). The expression of PSD-95mRNA in the MD group at different developmental stages was significantly reduced, and the relative expression was in MD. Group P45 was 0.470 + 0.005 (P450.01), which was 0.350 + 0.005 (P900.01) in group MDP90, and there was a significant difference in the relative expression of PSD-95mRNA between different stages of development in MD group (P0.05).
conclusion
1 in the critical period of visual development, visual experience regulates the expression of PSD-95 by regulating synaptic connections among visual neurons, and PSD-95 is one of the important molecular biological bases of the pathogenesis of amblyopia.
2 abnormal visual experience can influence the expression of PSD-95 in the visual cortex of adult rats. It is speculated that the neurons in the visual cortex of adult rats still have synaptic plasticity.
3 compared with the normal developmental rats, the expression of PSD-95 in the visual cortex of normal adult rats decreased, and the age factor was one of the factors affecting the expression of PSD-95.
【学位授予单位】：新乡医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R777.44

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