小鼠青光眼模型视网膜神经节细胞损伤与CD3ζ关系的实验研究
发布时间：2019-05-29 17:36
【摘要】：研究背景青光眼是世界上最主要的不可逆性致盲性眼病之一,引起周边视野的缺损直至中心视力消失。视网膜神经节细胞(retinal ganglion cell, RGC)的凋亡是青光眼的最主要特征之一,但是RGC损伤的具体机制至今不明,如何减少青光眼RGC的损害是世界性难题,观察RGC丢失的速度、形态的变化对揭示青光眼的病程有重要的临床指导意义。传统的研究RGC损伤的方法无法活体动态监测RGC的变化,目前国际上共焦激光显微镜活体观察转基因小鼠带自发荧光的RGC变化是一种新的趋势,但是主要集中在观察RGC数量的变化,最近有研究表明活体观察猴和小鼠RGC树突的可能性,但是还没有研究涉及到病理状态下,对RGC形态变化的观察。眼内压(introcular pressure, IOP)升高是青光眼发病的主要原因之一,但不能解释青光眼发病的所有特征。近年来免疫系统参与青光眼发病的相关研究越来越受关注。我们之前的研究已表明CD3δ是影响RGC树突形态和功能的关键因子,CD3δ-/-小鼠的树突密度明显大于同龄野生型小鼠。ShRNA沉默CD3δ在RGC的表达会导致该细胞树突明显增加,CD3ζ影响了生理状态下RGC树突的形态和功能,病理状态下是否导致了RGC的损伤有待进一步研究。目的 建立青光眼动物模型,选择合适的模型活体观察RGC变化,构建mCherry CD3ζshRNA表达载体,研究CD3ζ与RGC损伤的关系,探讨RNAi技术在视神经保护中的运用效果。 方法 1三种青光眼模型的建立：1)视神经挫伤模型：用能自动闭合的镊子夹伤视神经。2) NMDA诱导的视网膜损伤模型：玻璃体腔内注射NMDA (40nmol)导致RGC凋亡3)慢性高眼压模型：前房内注射微珠(10x 106 microbeads/ml)堵塞前房角导致眼压升高。 2 RGC损伤的观察：用共焦激光显微镜活体动态观察Thy1-CFP小鼠带自发荧光的RGC的数量变化和和Thy1-YFP小鼠带自发荧光的RGC的形态变化。 3 mCherry CD3ζshRNA表达载体构建：mCherry CD3ζshRNA表达载体是由编码shRNA的引物序列插入BamHI和XhoI位点之间。 4视网膜组织中CD3ζ的mRNA及蛋白表达水平检测：RT-PCR和Western blot法检测视网膜组织中CD3ζ的mRNA及蛋白表达水平。 5 Brn3b阳性的RGC检测：将mCherry CD3ζshRNA注射入小鼠玻璃体腔后,建立青光眼模型,取出视网膜行全视网膜铺片,计数Brn3b阳性的RGC。 结果 1建立了三种青光眼模型：1)视神经损伤模型2) NMDA诱导的视网膜损伤模型3)慢性高眼压模型 2视神经挫伤模型RGC减少主要集中于术后一周,7天时RGC减少97.4%(P＜0.001),NMDA诱导的RGC减少主要集中于术后一天,24小时RGC减少94.7%(P0.001),慢性高眼压导致的RGC减少持续缓慢,第4周RGC减少26.7%(P0.001)。 3用Thy1-CFP小鼠和Thy1-YFP小鼠建立慢性高眼压模型,活体观察结果显示,Thy1-CFP小鼠3周后RGC减少21%±4.8%(P0.001),6周后RGC减少30%±4.7%(P0.001), Thy1-YFP小鼠的RGC的树突丢失早于胞体消失。 4成功构建了mCherry-CD3ζshRNA的表达载体,CD3ζshRNA减少HEK293的70%的CD3ζ表达。 5三种青光眼模型视网膜组织中CD3ζ的mRNA表达上升(P0.05),CD3ζ蛋白质表达水平与正常视网膜相比没有明显变化。 6玻璃体腔注射mCherry CD3ζshRNA的三种青光眼模型中RGC的丢失没有明显减少(P0.05)。 结论 1视神经挫伤模型引起的RGC减少主要集中在术后一周,NMDA诱导的RGC减少主要集中于术后24小时,而前房内注射微珠堵塞房角诱发的高眼压模型的RGC减少是持续缓慢的,更加拟合临床青光眼的状态。 2可利用共焦激光显微镜活体观察已建立慢性高眼压模型的Thy1-CFP小鼠的RGC的数量减少和Thy1-YFP小鼠的RGC的树突丢失。 3视神经挫伤模型、NMDA诱导的视网膜损伤模型、慢性高眼压模型的视网膜组织中CD3ζ的mRNA水平表达上升,但CD3ζ的蛋白表达水平无明显变化,玻璃体腔注射mCherry CD3ζshRNA的的三种青光眼模型,RGC的损伤没有明显减少,提示CD3ζ能与RGC的损伤无关。
[Abstract]:The study of the background of glaucoma is one of the world's most important irreversibility-causing blindness, which causes the peripheral vision to be lost until the central vision disappears. The apoptosis of the retinal ganglion cell (RGC) is one of the most important features of the glaucoma, but the specific mechanism of the RGC injury is still unknown, and how to reduce the damage of the RGC in the glaucoma is a worldwide problem, and the rate of the loss of the RGC is observed. The change of morphology is of great clinical significance to reveal the course of glaucoma. The traditional method for studying the RGC injury can not dynamically monitor the change of the RGC in the living body, and the current international confocal laser microscope in-vivo observation of the RGC change of the spontaneous fluorescence of the transgenic mouse is a new trend, but mainly focuses on the observation of the change of the number of the RGC, Recent studies have shown the possibility of living in vivo observation of the RGC dendrites of monkeys and mice, but no studies have been conducted to investigate the changes in the form of RGC in the pathological state. The increase of intraocular pressure (IOP) is one of the main causes of the pathogenesis of glaucoma, but it is not possible to explain all the characteristics of glaucoma. In recent years, the involvement of the immune system in the pathogenesis of glaucoma has become more and more concerned. Our previous studies have shown that CD3 + is a key factor that affects the dendritic morphology and function of the RGC, and the dendritic density of CD3 +-/-mice is significantly greater than that of the wild-type mice of the same age. The expression of the RNA-silent CD3 antigen in the RGC leads to a significant increase in the dendritic growth of the cell, which affects the morphology and function of the RGC dendrites in the physiological state, and whether the damage to the RGC in the pathological state is to be further studied. Purpose To establish an animal model of glaucoma, to select a suitable model to observe the changes of RGC, to construct mCherry CD3/ shRNA expression vector, to study the relationship between CD3 antigen and RGC injury, and to explore the application of RNAi in the protection of optic nerve. effect . Method 1 Establishment of three glaucoma models:1) Optic nerve contusion model: the optic nerve is clipped with an automatic closed forceps.2) NMDA-induced retinal injury model: the injection of NMDA (40 nmol) in the vitreous cavity leads to the apoptosis of RGC 3. ) Chronic high intraocular pressure model: anterior chamber injection microbeads (10 x 106 microbeads/ ml) The changes in the number of spontaneous fluorescence of Thyl-CFP mice and the spontaneous emission of Thy1-YFP mice were observed by confocal laser microscope in vivo. The morphology of the fluorescent RGC was changed.3 mCherry CD3/ shRNA expression vector was constructed: mCherry CD3-shRNA expression vector was inserted by a primer sequence encoding shRNA RT-PCR and Western blot for the detection of retinal tissue. The mRNA and protein expression level of CD3 + in the middle and the expression level of the protein.5 Brn3b-positive RGC detection: after the mChierry CD3/ shRNA was injected into the vitreous cavity of the mouse, a glaucoma model was established and the retinal line was taken out. full view The results 1 established three glaucoma models:1) optic nerve injury model 2) NMDA-induced retinal injury model 3) chronic high-tension model 2, optic nerve contusion model RGC reduced main set RGC decreased by 97.4% (P <0.001) at 7 days after operation, and the decrease of RGC induced by NMDA was mainly concentrated on one day after operation, and the RGC decreased by 94.7% (P0.001) in 24 hours, and the RG induced by chronic high intraocular pressure C decreased by 26.7% (P0.001) in the fourth week, and the chronic high intraocular pressure model was established by Thyl-CFP mice and Thy1-YFP mice. In vivo, the RGC decreased by 21% and 4.8% after 3 weeks in Th1-CFP mice (P0.001), and the RGC decreased by 30% and 4.7% after 6 weeks (P 0.001), the dendritic loss of the RGC in the Thy1-YFP mice was early in the cell body, and the mCherry-CD3/ shRN was successfully constructed. A. The expression vector of CD3 + shRNA reduced the expression of CD3 antigen of 70% of HEK293. The mRN of CD3 + in the retina of three glaucoma models A. The expression level of CD3 + protein was not significantly changed as compared with that of normal retina (P0.05). er The loss of RGC in the three glaucoma models of dry CD3-shRNA was not significantly reduced (P0.05). At the time of, the decrease in the RGC of the high intraocular pressure model induced by the injection of microbeads in the anterior chamber was slow and more fitting to the state of the clinical glaucoma. Reduction of the number of RGC in Th1-CFP mice with chronic high intraocular pressure model and dendritic loss of the RGC in Th1-YFP mice.3. Optic nerve contusion model, NMDA-induced retinal injury model, and the mRNA level of CD3 in the retina of the chronic high-pressure model. Up to now, there was no significant change in the level of protein expression in CD3 +, and the injection of mC in the vitreous cavity
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R775
本文编号：2488093
[Abstract]:The study of the background of glaucoma is one of the world's most important irreversibility-causing blindness, which causes the peripheral vision to be lost until the central vision disappears. The apoptosis of the retinal ganglion cell (RGC) is one of the most important features of the glaucoma, but the specific mechanism of the RGC injury is still unknown, and how to reduce the damage of the RGC in the glaucoma is a worldwide problem, and the rate of the loss of the RGC is observed. The change of morphology is of great clinical significance to reveal the course of glaucoma. The traditional method for studying the RGC injury can not dynamically monitor the change of the RGC in the living body, and the current international confocal laser microscope in-vivo observation of the RGC change of the spontaneous fluorescence of the transgenic mouse is a new trend, but mainly focuses on the observation of the change of the number of the RGC, Recent studies have shown the possibility of living in vivo observation of the RGC dendrites of monkeys and mice, but no studies have been conducted to investigate the changes in the form of RGC in the pathological state. The increase of intraocular pressure (IOP) is one of the main causes of the pathogenesis of glaucoma, but it is not possible to explain all the characteristics of glaucoma. In recent years, the involvement of the immune system in the pathogenesis of glaucoma has become more and more concerned. Our previous studies have shown that CD3 + is a key factor that affects the dendritic morphology and function of the RGC, and the dendritic density of CD3 +-/-mice is significantly greater than that of the wild-type mice of the same age. The expression of the RNA-silent CD3 antigen in the RGC leads to a significant increase in the dendritic growth of the cell, which affects the morphology and function of the RGC dendrites in the physiological state, and whether the damage to the RGC in the pathological state is to be further studied. Purpose To establish an animal model of glaucoma, to select a suitable model to observe the changes of RGC, to construct mCherry CD3/ shRNA expression vector, to study the relationship between CD3 antigen and RGC injury, and to explore the application of RNAi in the protection of optic nerve. effect . Method 1 Establishment of three glaucoma models:1) Optic nerve contusion model: the optic nerve is clipped with an automatic closed forceps.2) NMDA-induced retinal injury model: the injection of NMDA (40 nmol) in the vitreous cavity leads to the apoptosis of RGC 3. ) Chronic high intraocular pressure model: anterior chamber injection microbeads (10 x 106 microbeads/ ml) The changes in the number of spontaneous fluorescence of Thyl-CFP mice and the spontaneous emission of Thy1-YFP mice were observed by confocal laser microscope in vivo. The morphology of the fluorescent RGC was changed.3 mCherry CD3/ shRNA expression vector was constructed: mCherry CD3-shRNA expression vector was inserted by a primer sequence encoding shRNA RT-PCR and Western blot for the detection of retinal tissue. The mRNA and protein expression level of CD3 + in the middle and the expression level of the protein.5 Brn3b-positive RGC detection: after the mChierry CD3/ shRNA was injected into the vitreous cavity of the mouse, a glaucoma model was established and the retinal line was taken out. full view The results 1 established three glaucoma models:1) optic nerve injury model 2) NMDA-induced retinal injury model 3) chronic high-tension model 2, optic nerve contusion model RGC reduced main set RGC decreased by 97.4% (P <0.001) at 7 days after operation, and the decrease of RGC induced by NMDA was mainly concentrated on one day after operation, and the RGC decreased by 94.7% (P0.001) in 24 hours, and the RG induced by chronic high intraocular pressure C decreased by 26.7% (P0.001) in the fourth week, and the chronic high intraocular pressure model was established by Thyl-CFP mice and Thy1-YFP mice. In vivo, the RGC decreased by 21% and 4.8% after 3 weeks in Th1-CFP mice (P0.001), and the RGC decreased by 30% and 4.7% after 6 weeks (P 0.001), the dendritic loss of the RGC in the Thy1-YFP mice was early in the cell body, and the mCherry-CD3/ shRN was successfully constructed. A. The expression vector of CD3 + shRNA reduced the expression of CD3 antigen of 70% of HEK293. The mRN of CD3 + in the retina of three glaucoma models A. The expression level of CD3 + protein was not significantly changed as compared with that of normal retina (P0.05). er The loss of RGC in the three glaucoma models of dry CD3-shRNA was not significantly reduced (P0.05). At the time of, the decrease in the RGC of the high intraocular pressure model induced by the injection of microbeads in the anterior chamber was slow and more fitting to the state of the clinical glaucoma. Reduction of the number of RGC in Th1-CFP mice with chronic high intraocular pressure model and dendritic loss of the RGC in Th1-YFP mice.3. Optic nerve contusion model, NMDA-induced retinal injury model, and the mRNA level of CD3 in the retina of the chronic high-pressure model. Up to now, there was no significant change in the level of protein expression in CD3 +, and the injection of mC in the vitreous cavity
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R775
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