本文選題：青光眼 + 動物模型　； 參考：《武漢大學(xué)》2016年博士論文

【摘要】：第一部分NMDA誘導(dǎo)小鼠視網(wǎng)膜興奮毒性損傷模型的特征目的：研究N-甲基-D-天冬氨酸(N-methyl-D-aspartate, NMDA)誘導(dǎo)視網(wǎng)膜興奮毒性損傷小鼠模型的形態(tài)和功能特征。方法：6周齡C57BL/6J小鼠隨機分為低、中、高三個劑量組,玻璃體腔內(nèi)分別注射NMDA10、20、30nmol,建立視網(wǎng)膜興奮損傷動物模型。我們在整體動物水平通過眼底照相、視網(wǎng)膜電圖(electroretinogram, ERG);在組織學(xué)水平通過視網(wǎng)膜鋪片熒光血管染色、視網(wǎng)膜石蠟切片HE染色、視神經(jīng)甲苯胺藍染色、全視網(wǎng)膜鋪片Brn-3a免疫熒光染色及RGCs計數(shù)觀察術(shù)后6h、12h、24h、48h、7dRGCs的損傷情況。結(jié)果：NMDA干預(yù)后24 h,RGCs數(shù)量明顯減少,IPL厚度變薄。眼底視網(wǎng)膜呈蒼白色改變,血管迂曲痙攣。Isolectin IB4熒光染色顯示視網(wǎng)膜血管,觀察到MDA造模后,24h內(nèi)視網(wǎng)膜血管結(jié)構(gòu)未見明顯損傷。ERG顯示a波和b波潛伏期的變化不大,b波振幅下降。光鏡下可見RGCs密度逐漸降低；視神經(jīng)軸索呈退行性變,與NMDA的作用時間呈正相關(guān)。RGCs計數(shù)顯示NMDA誘導(dǎo)的RGC減少主要集中于術(shù)后一天,24 h RGCs數(shù)量減少83.97%(P0.01)。結(jié)論：玻璃體腔內(nèi)注射NMDA可誘導(dǎo)RGCs的顯著減少和視網(wǎng)膜功能損害,與急性青光眼性損傷具有相似性,可為視神經(jīng)保護研究提供研究條件。檢測ERG b波,對于診斷和評估視網(wǎng)膜興奮損傷具有一定的意義。第二部分NMDA誘導(dǎo)視網(wǎng)膜視桿雙極細胞功能性PKCa轉(zhuǎn)運的機制研究目的：觀察NMDA誘導(dǎo)的視網(wǎng)膜興奮毒性損傷小鼠模型中視桿-雙極細胞中重要的功能性PKCa轉(zhuǎn)運現(xiàn)象并探討其機制。方法：6/3周齡C57BL/6J小鼠玻璃體腔內(nèi)注射NMDA30 nmol,建立視網(wǎng)膜興奮損傷動物模型。分別在損傷后12 h、24 h、48 h取出視網(wǎng)膜,利用視網(wǎng)膜切片免疫熒光及TUNEL凋亡染色,觀察視網(wǎng)膜的分層以及細胞存活情況、PKCa標(biāo)記的視桿-雙極細胞及NMDA受體各亞基在視網(wǎng)膜上的突觸表達；利用全細胞膜片鉗技術(shù),記錄視網(wǎng)膜各類型雙極細胞的電生理特性改變；通過單細胞PCR方法,檢測視桿-雙極細胞中mGluR6-Go-TRPMl信號通路相關(guān)組成和調(diào)控因子的mRNA水平。結(jié)果：觀察到NMDA在誘導(dǎo)內(nèi)層視網(wǎng)膜RGCs急性凋亡的同時,視桿-雙極細胞內(nèi)出現(xiàn)功能性PKCa轉(zhuǎn)運的現(xiàn)象；證實與外叢狀層上NR1、NR2B、NR2D亞基的突觸表達量明顯升高存在空間一致性。利用全細胞膜片鉗技術(shù),揭示了NMDA可特異性誘導(dǎo)視桿-雙極細胞mGluR6的功能變化,對光反應(yīng)消失,且損傷具有可逆性。進一步通過單細胞PCR檢測,NMDA造模后GDIs的上調(diào)和部分GAPs、RGS的下調(diào),共同導(dǎo)致了Gao亞基的失活,TRPM通道開放。結(jié)論：玻璃體腔內(nèi)注射NMDA,可誘導(dǎo)急性的神經(jīng)節(jié)細胞凋亡及雙極細胞損傷。通過作用mGluR6-Go-TRPM1這條信號通路,影響視桿雙極細胞的生理功能。相關(guān)機制研究可對今后治療人類視桿-雙極細胞機制相關(guān)的眼病起到指導(dǎo)性作用。
[Abstract]:Part one: the characteristics of NMDA induced retinal excitotoxicity injury model in mice objective: to study the morphological and functional characteristics of the model induced by N-methyl-D-aspartate (NMDA-N-methyl-D-aspartate) -induced retinal excitotoxicity injury in mice. Methods C57BL/6J mice at 6 weeks of age were randomly divided into three groups: low, middle and high dose groups, and intravitreous injection of NMDA1020 + 30nmol into the vitreous cavity to establish the animal model of retinal excitatory injury. At the overall animal level, we take pictures of the fundus, electroretinograms, ERGs; at the histological level, they are stained by fluorescent vessels on the retina, paraffin sections of the retina are stained with HE, and the optic nerves are stained with toluidine blue. Brn-3a immunofluorescence staining and RGCs count were used to observe the injury of RGCs at 6 h, 12 h, 24 h, 48 h and 7 d after operation. Results the number of RGCs in RGCs decreased significantly 24 h after the intervention of 1: NMDA, and the thickness of IPL became thinner. The retinal fundus showed pale white changes, vasospasm. Isolectin IB4 fluorescence staining showed retinal vessels. No obvious damage to retinal vascular structure was observed within 24 hours after MDA modeling. Under the light microscope, the density of RGCs decreased gradually, and the optic axons showed degenerative change, which was positively correlated with the action time of NMDA. RGCs count showed that the decrease of RGC induced by NMDA was mainly focused on the reduction of the number of RGCs at 24 h after operation by 83.97% (P 0.01). Conclusion: intravitreal injection of NMDA can significantly reduce RGCs and damage retinal function, which is similar to that of acute glaucoma injury, and may provide research conditions for the study of optic nerve protection. The detection of ERG b wave is of significance for the diagnosis and evaluation of retinal excitatory injury. The second part is the mechanism of functional PKCa transport in retinal rod bipolar cells induced by NMDA. Objective: to observe the important functional PKCa transport in the rod bipolar cells in the mouse model of retinal excitotoxicity injury induced by NMDA and to explore its mechanism. Methods NMDA30 nmol was injected into the vitreous of 6 / 3 week old C57BL/6J mice to establish the animal model of retinal excitatory injury. The retina was removed at 12 h, 24 h and 48 h after injury. The retinal sections were stained with immunofluorescence and TUNEL apoptosis. The synaptic expression of PKCa labeled optic rod bipolar cells and NMDA receptor subunits on the retina was observed, and the electrophysiological characteristics of various types of bipolar cells in the retina were recorded by whole-cell patch clamp technique. Single cell PCR method was used to detect the relative components of mGluR6-Go-TRPMl signaling pathway and the mRNA level of regulatory factors in rod-bipolar cells. Results: while NMDA induced acute apoptosis of RGCs in the inner layer of retina, functional PKCa transport was observed in rod bipolar cells, which was confirmed to be spatially consistent with the increase of synaptic expression of NR1, NR2BnR2BnR2D subunit in the outer plexiform layer. By using whole-cell patch clamp technique, it was revealed that NMDA could specifically induce the functional changes of mGluR6 in rod and bipolar cells, and the photoreaction disappeared and the damage was reversible. Furthermore, the up-regulation of GDIs and the down-regulation of some GAPs were detected by single cell PCR, which resulted in the opening of inactivated Gao subunit. Conclusion: intravitreal injection of NMDA can induce acute ganglion cell apoptosis and bipolar cell injury. The physiological function of rod bipolar cells is affected by mGluR6-Go-TRPM1 signaling pathway. The study of the related mechanism may play a guiding role in the treatment of ocular diseases related to the mechanism of human optic rod-bipolar cell.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R775;R-332

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