RCS大鼠視網(wǎng)膜重構中谷氨酸變化對神經(jīng)節(jié)細胞的影響研究
發(fā)布時間：2019-06-18 18:45
【摘要】：目的 通過研究RCS大鼠視網(wǎng)膜重構過程中谷氨酸的釋放、攝取和轉(zhuǎn)化各階段的變化規(guī)律，以及谷氨酸作為神經(jīng)遞質(zhì)的輸出改變對視網(wǎng)膜節(jié)細胞功能的影響，進而揭示視網(wǎng)膜變性過程中谷氨酸改變對視網(wǎng)膜神經(jīng)節(jié)細胞存活的影響，以及變性過程中視網(wǎng)膜神經(jīng)節(jié)細胞電生理功能的改變，為視網(wǎng)膜變性后神經(jīng)元的保護、視功能挽救和重建提供線索，為視網(wǎng)膜重構的基礎研究和臨床治療研究提供理論依據(jù)。 方法 1、采用免疫組織化學技術研究重構視網(wǎng)膜中蛋白激酶C的α亞單位(PKCα)和谷氨酰胺合成酶(GS)的分布；實時定量逆轉(zhuǎn)錄聚合酶鏈反應(RT-PCR)檢測不同時間點重構視網(wǎng)膜中PKCα與VGLUT-1mRNA表達量的變化規(guī)律； 2、Western免疫印跡技術檢測視網(wǎng)膜中GLAST蛋白和GS蛋白的表達水平； 3、膜片鉗技術檢測不同時間點視網(wǎng)膜神經(jīng)節(jié)細胞對谷氨酸的電生理反應性，最終闡明視網(wǎng)膜變性過程中，視網(wǎng)膜神經(jīng)節(jié)細胞的電生理功能的變化規(guī)律。 結果 1. RCS大鼠視網(wǎng)膜變性首先累及光感受器細胞，存活的視桿雙極細胞所占百分比隨變性發(fā)展逐漸增高；到視網(wǎng)膜變性晚期，內(nèi)核層和節(jié)細胞層的細胞仍有大量殘留。 2. RCS大鼠視網(wǎng)膜變性發(fā)展到中后期，作為神經(jīng)遞質(zhì)的谷氨酸釋放逐漸增多，但Müller細胞攝取谷氨酸的能力并不相應地增加，只是Müller細胞內(nèi)對谷氨酸的轉(zhuǎn)化能力逐漸增強；到視網(wǎng)膜變性晚期，谷氨酸的釋放量相對減少。 3.正常大鼠視網(wǎng)膜神經(jīng)節(jié)細胞鉗制在-80mV水平下受光刺激后誘發(fā)的內(nèi)向電流包括三種成份，即AMPA受體成份，GABA受體成份，誘發(fā)動作電位后產(chǎn)生的Na＋內(nèi)流成份；在此鉗制電壓下，視網(wǎng)膜神經(jīng)節(jié)細胞主要通過AMPA受體和GABA受體接受上級神經(jīng)元的輸入；并且內(nèi)向電流強度隨年齡增加無明顯改變，穩(wěn)定在一定水平，且谷氨酸電流強度也無明顯改變；NBQX不影響視網(wǎng)膜神經(jīng)節(jié)細胞內(nèi)向電流的各時程。 4. RCS大鼠視網(wǎng)膜中存活的對光刺激無反應的視網(wǎng)膜神經(jīng)節(jié)細胞仍保留了部分電生理功能；在視網(wǎng)膜變性早期，部分視網(wǎng)膜節(jié)細胞出現(xiàn)了功能障礙，光刺激信號不能通過上級神經(jīng)元傳遞到視網(wǎng)膜神經(jīng)節(jié)細胞；到視網(wǎng)膜變性晚期，存活的所有類型的視網(wǎng)膜節(jié)細胞皆接受不到上級神經(jīng)元的信號輸入。 5. RCS大鼠視網(wǎng)膜變性早期時就導致ON型視網(wǎng)膜節(jié)細胞接受的視網(wǎng)膜內(nèi)信息延遲、接受并處理信息的能力下降、視網(wǎng)膜信息輸出時間延遲，但對OFF型和ON-OFF型視網(wǎng)膜神經(jīng)節(jié)細胞無影響。但是，隨著視網(wǎng)膜變性的發(fā)展，各類型節(jié)細胞視網(wǎng)膜內(nèi)信息傳遞逐漸完全中斷，到晚期無視網(wǎng)膜信號輸出。 6. RCS大鼠視網(wǎng)膜神經(jīng)節(jié)細胞接受和處理的信息量較正常大鼠明顯減少了，，且這些信息主要是由視網(wǎng)膜神經(jīng)節(jié)細胞突觸后興奮性電流所介導的。 結論 1. RCS大鼠視網(wǎng)膜變性晚期仍有大量內(nèi)核層和節(jié)細胞層細胞殘留，為光感受器移植、植入視覺假體等治療方式恢復變性視網(wǎng)膜的視功能提供了實驗和理論基礎。 2. RCS大鼠視網(wǎng)膜變性中期游離的谷氨酸可能發(fā)生蓄積，到視網(wǎng)膜變性晚期神經(jīng)視網(wǎng)膜減少了對下級神經(jīng)元固有的谷氨酸能驅(qū)動，為“神經(jīng)元傳入性信號輸入的喪失導致了視網(wǎng)膜重構”的推測提供了重要的證據(jù)。 3.隨RCS大鼠視網(wǎng)膜變性發(fā)展，視網(wǎng)膜神經(jīng)節(jié)細胞逐漸出現(xiàn)功能障礙，不能接受上級神經(jīng)元的信號輸入，也無視網(wǎng)膜信號輸出；但存活的對光刺激無反應的視網(wǎng)膜神經(jīng)節(jié)細胞仍保留了部分電生理功能，這為視網(wǎng)膜重構后視功能的挽救，提供了理論和實驗基礎。
[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,
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：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,
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R774.1
【參考文獻】
相關期刊論文 前2條
1 陳麗峰;陳中山;曾玉曉;翁傳煌;陰正勤;;RCS大鼠視網(wǎng)膜色素變性過程中視網(wǎng)膜形態(tài)學研究[J];第三軍醫(yī)大學學報;2006年10期
2 李樹寧,王z牖
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