降解大鼠视皮层硫酸软骨素对NR2A和NR2B发育表达影响的研究
发布时间：2018-08-28 15:06
【摘要】： 在外界环境刺激和内在基因调控下,视觉系统可以发生形态和功能的适应性改变,这种发育的敏感时期称为视觉发育可塑性关键期(Critical period, CP)。在生后早期的关键期内,视觉经验可以促进突触之间的连接及中枢神经元网络系统的成熟。异常视觉环境下,如斜视、上睑下垂或先天性白内障等的存在,使儿童视网膜缺乏正常清晰的图像刺激而导致矫正视力低于正常值。弱视患者还往往伴有立体视觉、运动、知觉等的损害。如果在可塑性关键期内及时排除这些影响,弱视可以治愈,而在可塑性关键期终止以后,即使获得正常的视觉输入,弱视的治愈机率也会很低,因为,人类的视皮层可塑性随着关键期的终止被抑制了。在临床上,成年和大于12岁的儿童弱视患者基本不能被治愈。已经证明,弱视的发病部位位于视皮层。所以,如何启动被抑制的视皮层可塑性,是解决问题的关键。 新近研究发现,对视觉发育可塑性关键期终止有重要影响的因素是:视皮层内突触可塑性、局部兴奋性/抑制性神经元回路的成熟、神经元细胞外基质(extracellular matrix, ECM)、神经营养因子以及一些相关基因的表达。细胞外基质中的一些成分,如硫酸软骨素粘多糖(Chondroitin solfate proteoglycans, CSPGs),在中枢神经系统发育晚期逐渐浓缩成高密度的格子状结构,形成包围神经元胞体和树突的神经元周围网络(Perineuronal nets, PNNs),将视皮层神经元完全套入其中。Pizzorusso等人利用硫酸软骨素酶(chondroitinaseABC, chABC)降解成年大鼠视皮层中的CSPGs,恢复了单眼视觉剥夺成年鼠视皮层眼优势柱可塑性,说明CSPGs在视皮层可塑性中发挥了强大的抑制作用。而且暗饲养可以导致视皮层内PNNs数目显著减少,表明CSPGs参与形成的PNNs介入了视觉可塑性关键期的终止。既往研究亦发现,视皮层内兴奋性突触传递主要由N-甲基-D天冬氨酸受体(N-mehyl-D-aspartate, NMDA)及α-氨基-3羟基-5-甲基-4-异恶唑丙酸受体(a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, AMPA)介导。本实验室在之前的研究中,应用脑片膜片钳全细胞记录技术对大鼠视皮层进行研究发现:在正常大鼠可塑性关键期内,NMDA受体的作用相对于AMPA受体来说随着发育逐渐减弱,其中,NMDA受体介导的兴奋性突触传递特性与视皮层可塑性的变化相关。还有研究发现,NMDA受体亚单位NR2A( N-mehyl-D-aspartate 2A receptor, NR2A)、NR2B (N-mehyl-D-aspartate 2B receptor, NR2B)与视皮层可塑性密切相关,且NR2A/2B比率随发育逐渐增高,参与了视皮层可塑性的终止。通过形觉剥夺(Binocular form deprivation, BFD)或暗饲养可以使之出现降低,视皮层可塑性得到一定程度的恢复。 随着哺乳动物由幼年期向成熟期的发育,视皮层可塑性被抑制,同时,NMDA受体功能和CSPGs发育成熟均参与可塑性关键期的终止,两者之间可能具有相互联系。但是,降解视皮层CSPGs从而再激活视皮层可塑性的过程是否通过其对NMDA受体亚单位表达的影响来实现的呢?目前尚不清楚。多数研究集中在大鼠幼年期和成年期NR2A、NR2B的表达变化,而对于视皮层可塑性关键期终止前后这一时间段内NR2A、NR2B的表达和视皮层各层分布的研究甚少。为此,提出如下假设:在视皮层发育可塑性关键期终止前后,视皮层内NR2A、NR2B参与视皮层可塑性过程,降解大鼠视皮层细胞外基质中的CSPGs后,视皮层内NR2A、NR2B的表达发生变化。这一受体亚单位表达的变化可能是降解视皮层CSPGs恢复视皮层被抑制的可塑性的重要分子机制之一。为了验证此假说,我们从以下两个方面进行研究。 第一部分:视觉发育可塑性关键期终止前后正常大鼠视皮层NMDA受体亚单位NR2A和NR2B的表达 通过免疫荧光标记技术和蛋白质印迹技术,我们研究了出生后3到7周正常大鼠视皮层中NMDA受体亚单位NR2A、NR2B的发育变化。本部分研究结果发现:1.生后3-7周正常大鼠视皮层中NR2A定位在阳性细胞的胞膜和胞浆,在视皮层各层中均有明显表达,且在视皮层II-III层相对较集中分布。生后3-5周视皮层各层中阳性细胞数及总蛋白量逐渐增高(P0.05),5-7周增高不明显。2.生后3-7周正常大鼠视皮层中NR2B定位在阳性细胞的胞膜和胞浆,在视皮层各层中均有明显表达,但在各层间的分布并无明显差异;且在生后3周时各层阳性细胞数及总蛋白量表达水平最高,在生后3-6周其表达明显降低(P0.05),6-7周表达趋近一较稳定水平。3.通过对视皮层各层NMDA受体NR2A和NR2B表达的阳性细胞数及蛋白免疫印迹结果进行分析得出,随大鼠年龄增长(生后3-7周),NR2A/2B比率逐渐增加,且在生后3-6周比率增加幅度有统计学意义(P0.05)。 第二部分:视觉发育可塑性关键期终止前后降解大鼠视皮层硫酸软骨素对NR2A和NR2B表达的影响 本实验参照Pizzorusso等的动物模型方法、利用免疫荧光标记和蛋白质印迹技术,率先研究可塑性关键期终止前后CSPGs降解大鼠视皮层中NMDA受体亚单位NR2A、NR2B表达的变化。研究结果发现:1.降解大鼠视皮层中的CSPGs后,与正常组大鼠同时间点比较,视皮层II-III、V-VI层中NR2A阳性细胞数在生后4-7周有显著性差异,而视皮层IV层在生后5-7周有显著性差异,同时蛋白免疫印迹检测也表明视皮层中NR2A总蛋白量在生后4-7周出现显著性降低(P0.05);2.降解大鼠视皮层中的CSPGs后,NR2B的阳性细胞数及蛋白表达水平在生后各时间点较正常组大鼠均无显著变化;3.降解大鼠视皮层中的CSPGs后,视皮层中NR2A/2B比率在生后4-7周较正常组大鼠同时间点表达比率出现降低,有显著性差异(P0.05)。 由上述结果初步得出以下结论:1.在视觉可塑性关键期终止前后,正常大鼠视皮层中NMDA受体亚单位NR2A和NR2B参与了视皮层可塑性变化的过程,而且随着大鼠生后周龄的增加(生后3-7周),两者比率逐渐升高, NR2A的表达逐渐取代了NR2B的主导地位。2.降解大鼠视皮层中的CSPGs后,NMDA受体亚单位NR2A/2B表达的比率在生后4-7周出现降低,并且以NR2A表达的减少为主,这一受体亚单位构成变化可能是降解视皮层CSPGs后恢复视皮层可塑性的重要分子机制之一。
[Abstract]:During the critical period of postnatal development, visual experience promotes synaptic connections and central neural network systems. Maturity. Abnormal visual conditions, such as strabismus, ptosis or congenital cataract, make the retina of children lack normal and clear image stimulation and lead to corrected visual acuity below normal. Amblyopia patients are often accompanied by stereopsis, movement, perception and other damage. If these effects are eliminated in a timely manner during the critical period of plasticity, weakness. Visual cortex plasticity in humans is suppressed with the termination of the critical period. Clinically, amblyopia in adults and older than 12 years of age is virtually incurable. It has been shown that the onset of amblyopia is limited. The location is located in the visual cortex. Therefore, how to start the inhibition of the plasticity of the visual cortex is the key to solve the problem.
Recent studies have found that the key factors affecting the termination of visual development plasticity are synaptic plasticity in the visual cortex, maturation of local excitatory/inhibitory neuronal circuits, expression of extracellular matrix (ECM), neurotrophic factors and some related genes, and some of the components of extracellular matrix. Chondroitin sulfate mucopolysaccharides (CSPGs), such as chondroitin solfate proteoglycans (CSPGs), gradually condense into high-density lattice-like structures in the late development of the central nervous system, forming perineuronal nets (PNNs), which surround the cell bodies and dendrites of neurons, and completely encapsulate the neurons in the visual cortex. Chondroitinase ABC (chABC) degraded CSPGs in the visual cortex of adult rats, restored the plasticity of dominant columns in the visual cortex of monocular deprived adult rats, suggesting that CSPGs played a powerful inhibitory role in the plasticity of visual cortex, and the number of PNNNs in the visual cortex was significantly reduced by dark feeding, suggesting that CSPGs participated in the formation of visual cortex. Previous studies have also shown that excitatory synaptic transmission in the visual cortex is mainly mediated by N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxalepropionic acid (AMPA). In previous studies, whole-cell patch-clamp recording techniques were used to study the visual cortex in rats. It was found that the role of NMDA receptors decreased with the development of AMPA receptors during the critical period of plasticity in normal rats. Other studies have found that NR2A (N-mehyl-D-aspartate 2A receptor, NR2A) and NR2B (N-mehyl-D-aspartate 2B receptor, NR2B) are closely related to visual cortex plasticity, and NR2A/2B ratio increases with development, which participates in the termination of visual cortex plasticity. Feeding can make it appear lower, and the plasticity of the cortex is restored to some extent.
With the development of mammals from infancy to maturity, the plasticity of visual cortex is inhibited. At the same time, both NMDA receptor function and CSPGs are involved in the termination of the critical stage of plasticity, which may be interrelated. However, whether the process of degradation of visual cortex CSPGs and reactivation of visual cortex plasticity through its subunit to NMDA receptor may be related to each other. Most studies focused on the changes of NR2A and NR2B expression in young and adult rats, but few studies focused on the expression of NR2A and NR2B and the distribution of NR2B in the visual cortex before and after the termination of the critical period of visual cortex plasticity. NR2A and NR2B in the visual cortex participate in the plasticity process of the visual cortex before and after the termination of the critical period of plasticity. After the degradation of CSPGs in the extracellular matrix of the visual cortex, the expression of NR2A and NR2B in the visual cortex changes. The change of NR2A and NR2B expression may be an important molecular mechanism for the degradation of CSPGs in the visual cortex to restore the inhibited plasticity of the visual cortex. In order to test this hypothesis, we study the following two aspects.
Part I: Expression of NMDA receptor subunits NR2A and NR2B in the visual cortex of normal rats before and after termination of the critical period of visual plasticity
The development of NMDA receptor subunits NR2A and NR2B in the visual cortex of normal rats from 3 to 7 weeks after birth was studied by immunofluorescence labeling and Western blotting. The number of positive cells and total protein in each layer of visual cortex gradually increased at 3-5 weeks postnatal (P 0.05), but not at 5-7 weeks postnatal (P 0.05). 2. NR2B was localized in the membrane and cytoplasm of positive cells in the visual cortex of normal rats at 3-7 weeks postnatal, and was obviously expressed in all layers of visual cortex, but in different layers of visual cortex. There was no significant difference in the number of positive cells and the expression of total protein in different layers at 3 weeks after birth. The expression of positive cells and total protein decreased significantly at 3-6 weeks after birth (P 0.05), and reached a stable level at 6-7 weeks. The NR2A/2B ratio increased gradually with the age of rats (3-7 weeks after birth), and the increase of NR2A/2B ratio at 3-6 weeks after birth was statistically significant (P 0.05).
Part 2: Effect of chondroitin sulfate on the expression of NR2A and NR2B in visual cortex of rats before and after the termination of critical period of visual development plasticity
Referring to Pizzorusso's animal model, immunofluorescence labeling and protein imprinting were used to study the changes of the expression of NMDA receptor subunits NR2A and NR2B in the visual cortex of rats before and after the termination of the critical period of plasticity. The number of NR2A-positive cells in visual cortex II-III and V-VI was significantly different at 4-7 weeks after birth, while the number of NR2A-positive cells in visual cortex IV was significantly different at 5-7 weeks after birth. The total protein content of NR2A in visual cortex decreased significantly at 4-7 weeks after birth (P 0.05). The expression of NR2A/2B in the visual cortex was significantly lower than that in the normal group at 4-7 weeks after birth (P 0.05).
The results are as follows: 1. Before and after the termination of the critical period of visual plasticity, the NMDA receptor subunits NR2A and NR2B in the visual cortex of normal rats participated in the process of visual cortex plasticity, and the ratio of NR2A and NR2B increased gradually with the increase of postnatal age (postnatal 3-7 weeks). 2. After degradation of CSPGs in the visual cortex of rats, the expression ratio of NR2A/2B, a NMDA receptor subunit, decreased at 4-7 weeks postnatal, and the expression of NR2A was mainly decreased. The change of this receptor subunit composition may be one of the important molecular mechanisms of restoring visual cortex plasticity after degradation of CSPGs in the visual cortex.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R77
[Abstract]:During the critical period of postnatal development, visual experience promotes synaptic connections and central neural network systems. Maturity. Abnormal visual conditions, such as strabismus, ptosis or congenital cataract, make the retina of children lack normal and clear image stimulation and lead to corrected visual acuity below normal. Amblyopia patients are often accompanied by stereopsis, movement, perception and other damage. If these effects are eliminated in a timely manner during the critical period of plasticity, weakness. Visual cortex plasticity in humans is suppressed with the termination of the critical period. Clinically, amblyopia in adults and older than 12 years of age is virtually incurable. It has been shown that the onset of amblyopia is limited. The location is located in the visual cortex. Therefore, how to start the inhibition of the plasticity of the visual cortex is the key to solve the problem.
Recent studies have found that the key factors affecting the termination of visual development plasticity are synaptic plasticity in the visual cortex, maturation of local excitatory/inhibitory neuronal circuits, expression of extracellular matrix (ECM), neurotrophic factors and some related genes, and some of the components of extracellular matrix. Chondroitin sulfate mucopolysaccharides (CSPGs), such as chondroitin solfate proteoglycans (CSPGs), gradually condense into high-density lattice-like structures in the late development of the central nervous system, forming perineuronal nets (PNNs), which surround the cell bodies and dendrites of neurons, and completely encapsulate the neurons in the visual cortex. Chondroitinase ABC (chABC) degraded CSPGs in the visual cortex of adult rats, restored the plasticity of dominant columns in the visual cortex of monocular deprived adult rats, suggesting that CSPGs played a powerful inhibitory role in the plasticity of visual cortex, and the number of PNNNs in the visual cortex was significantly reduced by dark feeding, suggesting that CSPGs participated in the formation of visual cortex. Previous studies have also shown that excitatory synaptic transmission in the visual cortex is mainly mediated by N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxalepropionic acid (AMPA). In previous studies, whole-cell patch-clamp recording techniques were used to study the visual cortex in rats. It was found that the role of NMDA receptors decreased with the development of AMPA receptors during the critical period of plasticity in normal rats. Other studies have found that NR2A (N-mehyl-D-aspartate 2A receptor, NR2A) and NR2B (N-mehyl-D-aspartate 2B receptor, NR2B) are closely related to visual cortex plasticity, and NR2A/2B ratio increases with development, which participates in the termination of visual cortex plasticity. Feeding can make it appear lower, and the plasticity of the cortex is restored to some extent.
With the development of mammals from infancy to maturity, the plasticity of visual cortex is inhibited. At the same time, both NMDA receptor function and CSPGs are involved in the termination of the critical stage of plasticity, which may be interrelated. However, whether the process of degradation of visual cortex CSPGs and reactivation of visual cortex plasticity through its subunit to NMDA receptor may be related to each other. Most studies focused on the changes of NR2A and NR2B expression in young and adult rats, but few studies focused on the expression of NR2A and NR2B and the distribution of NR2B in the visual cortex before and after the termination of the critical period of visual cortex plasticity. NR2A and NR2B in the visual cortex participate in the plasticity process of the visual cortex before and after the termination of the critical period of plasticity. After the degradation of CSPGs in the extracellular matrix of the visual cortex, the expression of NR2A and NR2B in the visual cortex changes. The change of NR2A and NR2B expression may be an important molecular mechanism for the degradation of CSPGs in the visual cortex to restore the inhibited plasticity of the visual cortex. In order to test this hypothesis, we study the following two aspects.
Part I: Expression of NMDA receptor subunits NR2A and NR2B in the visual cortex of normal rats before and after termination of the critical period of visual plasticity
The development of NMDA receptor subunits NR2A and NR2B in the visual cortex of normal rats from 3 to 7 weeks after birth was studied by immunofluorescence labeling and Western blotting. The number of positive cells and total protein in each layer of visual cortex gradually increased at 3-5 weeks postnatal (P 0.05), but not at 5-7 weeks postnatal (P 0.05). 2. NR2B was localized in the membrane and cytoplasm of positive cells in the visual cortex of normal rats at 3-7 weeks postnatal, and was obviously expressed in all layers of visual cortex, but in different layers of visual cortex. There was no significant difference in the number of positive cells and the expression of total protein in different layers at 3 weeks after birth. The expression of positive cells and total protein decreased significantly at 3-6 weeks after birth (P 0.05), and reached a stable level at 6-7 weeks. The NR2A/2B ratio increased gradually with the age of rats (3-7 weeks after birth), and the increase of NR2A/2B ratio at 3-6 weeks after birth was statistically significant (P 0.05).
Part 2: Effect of chondroitin sulfate on the expression of NR2A and NR2B in visual cortex of rats before and after the termination of critical period of visual development plasticity
Referring to Pizzorusso's animal model, immunofluorescence labeling and protein imprinting were used to study the changes of the expression of NMDA receptor subunits NR2A and NR2B in the visual cortex of rats before and after the termination of the critical period of plasticity. The number of NR2A-positive cells in visual cortex II-III and V-VI was significantly different at 4-7 weeks after birth, while the number of NR2A-positive cells in visual cortex IV was significantly different at 5-7 weeks after birth. The total protein content of NR2A in visual cortex decreased significantly at 4-7 weeks after birth (P 0.05). The expression of NR2A/2B in the visual cortex was significantly lower than that in the normal group at 4-7 weeks after birth (P 0.05).
The results are as follows: 1. Before and after the termination of the critical period of visual plasticity, the NMDA receptor subunits NR2A and NR2B in the visual cortex of normal rats participated in the process of visual cortex plasticity, and the ratio of NR2A and NR2B increased gradually with the increase of postnatal age (postnatal 3-7 weeks). 2. After degradation of CSPGs in the visual cortex of rats, the expression ratio of NR2A/2B, a NMDA receptor subunit, decreased at 4-7 weeks postnatal, and the expression of NR2A was mainly decreased. The change of this receptor subunit composition may be one of the important molecular mechanisms of restoring visual cortex plasticity after degradation of CSPGs in the visual cortex.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R77
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