RCS大鼠视网膜重构中谷氨酸变化对神经节细胞的影响研究
发布时间：2019-06-18 18:45
【摘要】：目的 通过研究RCS大鼠视网膜重构过程中谷氨酸的释放、摄取和转化各阶段的变化规律，以及谷氨酸作为神经递质的输出改变对视网膜节细胞功能的影响，进而揭示视网膜变性过程中谷氨酸改变对视网膜神经节细胞存活的影响，以及变性过程中视网膜神经节细胞电生理功能的改变，为视网膜变性后神经元的保护、视功能挽救和重建提供线索，为视网膜重构的基础研究和临床治疗研究提供理论依据。 方法 1、采用免疫组织化学技术研究重构视网膜中蛋白激酶C的α亚单位(PKCα)和谷氨酰胺合成酶(GS)的分布；实时定量逆转录聚合酶链反应(RT-PCR)检测不同时间点重构视网膜中PKCα与VGLUT-1mRNA表达量的变化规律； 2、Western免疫印迹技术检测视网膜中GLAST蛋白和GS蛋白的表达水平； 3、膜片钳技术检测不同时间点视网膜神经节细胞对谷氨酸的电生理反应性，最终阐明视网膜变性过程中，视网膜神经节细胞的电生理功能的变化规律。 结果 1. RCS大鼠视网膜变性首先累及光感受器细胞，存活的视杆双极细胞所占百分比随变性发展逐渐增高；到视网膜变性晚期，内核层和节细胞层的细胞仍有大量残留。 2. RCS大鼠视网膜变性发展到中后期，作为神经递质的谷氨酸释放逐渐增多，但Müller细胞摄取谷氨酸的能力并不相应地增加，只是Müller细胞内对谷氨酸的转化能力逐渐增强；到视网膜变性晚期，谷氨酸的释放量相对减少。 3.正常大鼠视网膜神经节细胞钳制在-80mV水平下受光刺激后诱发的内向电流包括三种成份，即AMPA受体成份，GABA受体成份，诱发动作电位后产生的Na＋内流成份；在此钳制电压下，视网膜神经节细胞主要通过AMPA受体和GABA受体接受上级神经元的输入；并且内向电流强度随年龄增加无明显改变，稳定在一定水平，且谷氨酸电流强度也无明显改变；NBQX不影响视网膜神经节细胞内向电流的各时程。 4. RCS大鼠视网膜中存活的对光刺激无反应的视网膜神经节细胞仍保留了部分电生理功能；在视网膜变性早期，部分视网膜节细胞出现了功能障碍，光刺激信号不能通过上级神经元传递到视网膜神经节细胞；到视网膜变性晚期，存活的所有类型的视网膜节细胞皆接受不到上级神经元的信号输入。 5. RCS大鼠视网膜变性早期时就导致ON型视网膜节细胞接受的视网膜内信息延迟、接受并处理信息的能力下降、视网膜信息输出时间延迟，但对OFF型和ON-OFF型视网膜神经节细胞无影响。但是，随着视网膜变性的发展，各类型节细胞视网膜内信息传递逐渐完全中断，到晚期无视网膜信号输出。 6. RCS大鼠视网膜神经节细胞接受和处理的信息量较正常大鼠明显减少了，，且这些信息主要是由视网膜神经节细胞突触后兴奋性电流所介导的。 结论 1. RCS大鼠视网膜变性晚期仍有大量内核层和节细胞层细胞残留，为光感受器移植、植入视觉假体等治疗方式恢复变性视网膜的视功能提供了实验和理论基础。 2. RCS大鼠视网膜变性中期游离的谷氨酸可能发生蓄积，到视网膜变性晚期神经视网膜减少了对下级神经元固有的谷氨酸能驱动，为“神经元传入性信号输入的丧失导致了视网膜重构”的推测提供了重要的证据。 3.随RCS大鼠视网膜变性发展，视网膜神经节细胞逐渐出现功能障碍，不能接受上级神经元的信号输入，也无视网膜信号输出；但存活的对光刺激无反应的视网膜神经节细胞仍保留了部分电生理功能，这为视网膜重构后视功能的挽救，提供了理论和实验基础。
[Abstract]:Purpose The changes of glutamate release, uptake and transformation in various stages of the retinal remodeling of the RCS rats were studied, and the function of the retinal ganglion cells was changed as the output of the neurotransmitter. in response to that effect of glutamate change on the survival of the retinal ganglion cells during the degeneration of the retina, and the changes of the electrical physiological function of the retinal ganglion cells in the degeneration process, provide a line for the protection of the neurons after the degeneration of the retina, The theory of basic research and clinical treatment of retinal reconstruction is provided in this paper. It was reported. Method 1. Immunohistochemistry was used to study the expression of protein kinase C in the retina and the glutamate synthase (G) in the retina. Distribution of S); Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) to detect the expression of PKC and VGLUT-1 mRNA in the retina at different time points The changes of GLAST protein and GS in the retina were detected by Western immunoblotting. protein The electrophysiologic response of the retinal ganglion cells to the glutamate in different time points is detected by the patch clamp technique, and the electrical activity of the retinal ganglion cells during the retinal degeneration is finally clarified. physiology Results 1. The retinal degeneration of the RCS rats first involved the photoreceptor cells, and the percentage of the surviving visual rod bipolar cells increased with the development of the degeneration. In the middle and late stage of retinal degeneration of the RCS rats, the release of glutamic acid as a neurotransmitter gradually increased, but the ability of the M-ller cells to take the glutamic acid did not increase accordingly, but the transformation of the glutamic acid in the M-ller cells was only The ability to gradually increase; to the retina. 3. The inward current induced by the light-receiving stimulation at the level of-80 mV of the normal rat retinal ganglion cells included three components, namely, the AMPA receptor component, the GABA receptor component, the induced action potential post-production, Under the clamping voltage, the retinal ganglion cells are mainly input by the AMPA receptor and the GABA receptor, and the inward current intensity is not obviously changed with the age, and is stable to a certain extent There is no significant change in the current intensity of the glutamic acid, and the NBQX does not. In response to the time-course of the inward current in the retinal ganglion cells.4. The non-reactive retinal ganglion cells in the RCS rat's retina still retain some of the electrical physiological functions, and in the retina In the early stage, some of the retinal ganglion cells had dysfunction, and the light-stimulating signal could not pass through the superior neurons to the retinal ganglion cells; to the late stage of the degeneration of the retina, all types of visual networks that survived 5. In the early stage of retinal degeneration of the RCS rats, the on-the-retinal information of the on-type retinal ganglion cells was delayed, the ability to receive and process the information was decreased, and the retinal information output time delayed, but for the OF There is no effect on the F-and ON-OFF type of retinal ganglion cells. However, with the development of the degeneration of the retina, the intra-retinal letters of various types of cells In the RCS rats, the amount of information received and treated in the retinal ganglion cells of the RCS rats was significantly reduced and the information was if that's Conclusion 1. The retinal degeneration of the RCS rats still has a large number of core layers and cell layer cell residues in the late stage of degeneration. 2. The free glutamic acid in the middle of the retinal degeneration of the RCS rats may accumulate, and the late-stage retinal degeneration of the retina can reduce the inherent glutamate energy of the inferior neurons. 3. With the development of the retinal degeneration of the RCS rats, the retinal ganglion cells gradually function, and the signal input of the superior neurons can not be accepted, and the retinal signal is not output; but the surviving photostimulation of the non-reactive retinal ganglion cell still retains a portion of the cell,
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R774.1
[Abstract]:Purpose The changes of glutamate release, uptake and transformation in various stages of the retinal remodeling of the RCS rats were studied, and the function of the retinal ganglion cells was changed as the output of the neurotransmitter. in response to that effect of glutamate change on the survival of the retinal ganglion cells during the degeneration of the retina, and the changes of the electrical physiological function of the retinal ganglion cells in the degeneration process, provide a line for the protection of the neurons after the degeneration of the retina, The theory of basic research and clinical treatment of retinal reconstruction is provided in this paper. It was reported. Method 1. Immunohistochemistry was used to study the expression of protein kinase C in the retina and the glutamate synthase (G) in the retina. Distribution of S); Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) to detect the expression of PKC and VGLUT-1 mRNA in the retina at different time points The changes of GLAST protein and GS in the retina were detected by Western immunoblotting. protein The electrophysiologic response of the retinal ganglion cells to the glutamate in different time points is detected by the patch clamp technique, and the electrical activity of the retinal ganglion cells during the retinal degeneration is finally clarified. physiology Results 1. The retinal degeneration of the RCS rats first involved the photoreceptor cells, and the percentage of the surviving visual rod bipolar cells increased with the development of the degeneration. In the middle and late stage of retinal degeneration of the RCS rats, the release of glutamic acid as a neurotransmitter gradually increased, but the ability of the M-ller cells to take the glutamic acid did not increase accordingly, but the transformation of the glutamic acid in the M-ller cells was only The ability to gradually increase; to the retina. 3. The inward current induced by the light-receiving stimulation at the level of-80 mV of the normal rat retinal ganglion cells included three components, namely, the AMPA receptor component, the GABA receptor component, the induced action potential post-production, Under the clamping voltage, the retinal ganglion cells are mainly input by the AMPA receptor and the GABA receptor, and the inward current intensity is not obviously changed with the age, and is stable to a certain extent There is no significant change in the current intensity of the glutamic acid, and the NBQX does not. In response to the time-course of the inward current in the retinal ganglion cells.4. The non-reactive retinal ganglion cells in the RCS rat's retina still retain some of the electrical physiological functions, and in the retina In the early stage, some of the retinal ganglion cells had dysfunction, and the light-stimulating signal could not pass through the superior neurons to the retinal ganglion cells; to the late stage of the degeneration of the retina, all types of visual networks that survived 5. In the early stage of retinal degeneration of the RCS rats, the on-the-retinal information of the on-type retinal ganglion cells was delayed, the ability to receive and process the information was decreased, and the retinal information output time delayed, but for the OF There is no effect on the F-and ON-OFF type of retinal ganglion cells. However, with the development of the degeneration of the retina, the intra-retinal letters of various types of cells In the RCS rats, the amount of information received and treated in the retinal ganglion cells of the RCS rats was significantly reduced and the information was if that's Conclusion 1. The retinal degeneration of the RCS rats still has a large number of core layers and cell layer cell residues in the late stage of degeneration. 2. The free glutamic acid in the middle of the retinal degeneration of the RCS rats may accumulate, and the late-stage retinal degeneration of the retina can reduce the inherent glutamate energy of the inferior neurons. 3. With the development of the retinal degeneration of the RCS rats, the retinal ganglion cells gradually function, and the signal input of the superior neurons can not be accepted, and the retinal signal is not output; but the surviving photostimulation of the non-reactive retinal ganglion cell still retains a portion of the cell,
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R774.1
【参考文献】
相关期刊论文 前2条
1 陈丽峰;陈中山;曾玉晓;翁传煌;阴正勤;;RCS大鼠视网膜色素变性过程中视网膜形态学研究[J];第三军医大学学报;2006年10期
2 李树宁,王z牖
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