NMDA受体在大鼠和鲫鱼视网膜上的表达分布

发布时间：2018-03-19 04:27

本文选题：NMDA受体亚基1　切入点：双极细胞　出处：《武汉大学》2012年博士论文　论文类型：学位论文

【摘要】：目的：研究离子型谷氨酸受体NMDA (N-methyl-D-aspartate)受体的功能性亚基1(NMDAR1,NR1)蛋白在大鼠视网膜上的表达分布情况。方法：采用western blot技术对大鼠视网膜组织匀浆行免疫印迹,验证所用抗NR1抗体的特异性。用免疫组化荧光单标技术,在SD大鼠视网膜垂直冰冻切片上研究NR1蛋白在大鼠视网膜上的大致分布,又使用免疫荧光双标技术研究其在视网膜双极细胞(bipolar cell, BC)、无长突细胞(amacrine cell, AC)及神经节细胞(ganglion cell, GC)上的表达分布。结果：western blot分析结果显示本实验所用NR1抗体有较好的特异性。免疫荧光单标法结果显示NR1亚基的免疫阳性产物弥散分布在大鼠视网膜的外网层(outer plexiform layer, OPL)和内网层(inner plexiform layer, IPL)全层,且内核层(inner nuclear layer, INL)及神经节细胞层(ganglion cell layer, GCL)的不同种类细胞均有NR1蛋白的分布,但外核层(outer nuclear layer, ONL)没有观察到明显的NR1的阳性反应。进一步采用细胞特异性标记物和NR1抗体免疫双标,显示CHX10阳性的双极细胞胞体、PKCa阳性的视杆信号主导和视锥信号主导的ON型双极细胞(rod-dominant bipolar cell, RBC; cone-domidant ON type bipolar cell, ON-CBC)、recoverin阳性的ON-CBC和视锥信号主导的OFF型双极细胞(cone-dominant OFF type bipolar cell, OFF-CBC)和NR1均有共表达。我们观察到胆碱能、多巴胺能、γ-氨基丁酸能和甘氨酸能的无长突细胞均和NR1有共表达。用于标记神经节细胞的特异性标记物Bm3a阳性的细胞也有NR1蛋白的表达。结论：NMDA受体参与了外层及内层视网膜的信息传递。研究提供了NR1亚基参与ON型和OFF型双极细胞两条通路的证据,有力支持了NMDA受体在视网膜信号传导通路中的调节作用。NR1蛋白在神经节细胞的表达则表明NMDA受体也很可能参与青光眼或视网膜缺血性疾病中兴奋毒性导致的神经节细胞凋亡。目的：研究离子型谷氨酸受体NMDA受体的调节性亚基3A和3B(NMDAR3A-NMDAR3B, NR3A-NR3B)蛋白在鲫鱼视网膜上的表达分布情况。方法：采用western blot技术对鲫鱼视网膜组织匀浆行免疫印迹,验证所用抗NR3A和抗NR3B抗体的特异性。用免疫组化荧光单标及共聚焦激光扫描显微技术,在鲫鱼视网膜垂直冰冻切片上研究NR3A和NR3B蛋白在鲫鱼视网膜特别是外层视网膜上的表达分布。结果：western blot分析结果显示本实验所用抗NR3A和抗NR3B抗体分别较特异性地标记鲫鱼视网膜中的NR3A和NR3B蛋白。免疫荧光法结果显示NR3A的免疫阳性产物弥散分布在鲫鱼视网膜的外网层,呈点状或弥散状分布在内网层,内核层及神经节细胞层的不同种类细胞均有NR3A蛋白的分布。重点可见外核层细胞胞体、轴突和轴突终末上有NR3A的强阳性免疫反应。NR3B的免疫阳性产物弥散性分布在OPL和IPL,亦分布在INL和GCL少许细胞上,但阳性信号均比较弱。重点可见ONL只有部分光感受器细胞的胞体和轴突有较强的免疫阳性反应。结论：NR3A和NR3B蛋白的广泛表达进一步表明NMDA参与外层及内层视网膜的信息传递。NR3亚基在光感受器细胞的表达表明其可能参与年龄相关性黄斑变性和遗传性视网膜退行性疾病发病过程中兴奋毒性导致的光感受器细胞凋亡,其在神经节细胞层的表达则表明NR3亚基也可能参与青光眼或视网膜缺血性疾病中兴奋毒性导致的神经节细胞凋亡。
[Abstract]:Objective: To study the ionotropic glutamate receptor NMDA (N-methyl-D-aspartate) receptor functional subunit 1 (NMDAR1, NR1) protein expression in the rat retina distribution.
Methods: rat retina tissue homogenate by Western blotting using Western blot technology, the specificity of anti NR1 antibodies for validation. Immunohistochemical single labeled fluorescence technique in rat retina SD vertical frozen sections of the general distribution of NR1 protein in the rat retina, and using double immunofluorescence analysis the retinal bipolar cells (bipolar cell, BC), amacrine cells (amacrine, cell, AC) and ganglion cells (ganglion cell, GC) on the expression of the distribution.
Results: Western blot analysis showed that the experiment uses NR1 antibody has good specificity. Immunofluorescence single standard method showed that NR1 subunit immunoreactive products dispersed in the outer plexiform layer of rat retina (outer plexiform, layer, OPL) and the inner plexiform layer (inner plexiform layer, IPL) layer, and the kernel layer (inner nuclear layer, INL) and ganglion cell layer (ganglion cell layer, GCL) distribution of different types of cells were NR1 protein, but the outer nuclear layer (outer nuclear layer, ONL) observed no obvious NR1 positive reaction. The cell specific marker NR1 antibody and immunohistochemical staining showed. CHX10 positive bipolar cells, ON bipolar cells and rod signal leading cone signals as the dominant positive PKCa (rod-dominant bipolar cell cone-domidant ON type bipolar, RBC; cell, ON-CBC), recoverin + ON -CBC and cone signals leading OFF type bipolar cells (cone-dominant OFF type bipolar cell, OFF-CBC) and NR1 were co expression. We observed cholinergic, dopaminergic and GABAergic and glycinergic amacrine cells and NR1 were co expressed in ganglion cells. Marker specific marker Bm3a positive cells also expressed NR1 protein.
Conclusion: the NMDA receptors in the outer layer and the inner layer of the retina. The transmission of information provides the NR1 subunit in ON type and OFF type bipolar cells two pathways of evidence research, the strong support of the NMDA receptor in retinal signal transduction pathways in the regulation of.NR1 protein expression in ganglion cells showed that NMDA receptor is likely to participate in ganglion cell apoptosis induced glaucoma or retinal excited ischemic diseases.
Objective: To study the ionotropic glutamate receptor NMDA receptor regulatory subunit 3A and 3B (NMDAR3A-NMDAR3B, NR3A-NR3B) protein expression in the carp retina on the distribution.
Methods: the retinal tissue homogenate by Western blot using western carp blot technology, specific anti NR3A and anti NR3B antibodies for validation. Immunohistochemical single labeled fluorescence and confocal laser scanning microscopy, in the carp retina vertical freezing sections of NR3A and NR3B protein in the carp retina especially the expression and distribution of the outer layer on the retina.
Results: Western blot analysis showed that the anti NR3A and anti NR3B antibodies were specific markers in the carp retina NR3A and NR3B protein. Immunofluorescence assay showed that the immune positive products distributed in the outer plexiform layer of carp retinal NR3A, spotty or dispersion distribution in the inner plexiform layer, kernel distribution layer and ganglion cell layer in different cell types of NR3A protein. The visible outer nuclear layer cells, the strong immunoreactivity of.NR3B axons and axon terminal of NR3A immunoreactive products diffuse distribution in OPL and IPL, INL and GCL also distributed in a cell, but the positive signals were compared weak. ONL visible only part of photoreceptor cells in cell bodies and axons have strong immunoreactivity.
Conclusion: the expression of NR3A and NR3B protein are further suggested the involvement of NMDA in the outer and inner retinal information transfer.NR3 subunit expression in photoreceptor cells showed that it may be involved in age-related macular degeneration and hereditary retinal degenerative diseases in the pathogenesis of excited photoreceptor apoptosis toxicity caused by its expression in ganglion cell layer showed that the NR3 subunit may also be involved in ganglion cell apoptosis induced glaucoma or retinal excited ischemic diseases.

【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R774.1

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