【作者】 丁纯；

【导师】 唐罗生； 田宁；

【作者基本信息】 中南大学 ， 眼科， 2011， 博士

【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%(P<0.001),慢性高眼压导致的RGC减少持续缓慢,第4周RGC减少26.7%(P<0.001)。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%(P<0.001),6周后RGC减少30%±4.7%(P<0.001), 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(P<0.05),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(P>0.05)。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。更多还原

【Abstract】 Backgrouds Glaucoma is a leading cause of irreversible blindness in the world, resulting in an initial loss of peripheral vision with central visual defects occurring much later. The progressive death of retinal ganglion cell (RGC) is a key character of glaucoma.But the pathogenesis of the death of retinal ganglion cell is still unclear now.How to reduce the RGC damage of glaucoma is a hard problem.It is very meaningful to study the decrease speed and the morphology change of RGC.Confocal scanning laser microscopy is used to observe the RGC number changes of transgenic mice with expression of fluorescent protein in RGC under some pathological conditions.Two recent studies demonstrated the possibility of visualizing RGC dendrites in vivo in monkey and mouse.However,no study has provided in vivo data to determine the morphological changes of RGC. The increased intraocular pressure does not explain glaucoma in all patients but can be considered as a risk factor of the disease. People pay more and more attention to the relationship of immune system and glaucoma. Our recent study have presented strong evidence that the immune molecule CD3ζresulted in a significant change of RGC dendritic growth and elimination in developing retina.RGC dendritic density increased in mature CD3ζ-/-mice.RGC dendritic density increased After the expression of CD3ζdecreased in RGC by shRNA.CD3ζaffect the morphology and function of RGC dendrite under physiological condition.However,it is still unclear that whether CD3ζis related to the damage of RGCPurposes1 To establish three types of glaucoma model.2 To image RGC in vivo in the selected glaucoma model.3 To construct the mCherry CD3ζshRNA vector.4 To observe the effect of RNAi technique usage in retinal neuroprotection.Methods1 We set up three types of glaucoma model:1)optic nerve crush model:the optic nerve was clamped using a pair of self-closing tweezers.2)NMDA intravitreal injection model:Hamilton injector was used to inject NMDA(40nmol) into the vitreous body.3)chronic high intraocular pressure model:microbeads (10x106microbeads/ml) were injected into the anterior chamber to obstruct the angle of anterior chamber.2 A confocal scanning laser microscopy was used to image Thyl-CFP mice and Thyl-YFP mice to observe the damage of RGCs.3 The mCherry CD3ζshRNA expression constructs were generated by inserting a 68 bp shRNA template sequence between BamHI and XhoI sites.4 Real-time PCR and Western blot assy were used to detect CD3ζin retinal tissues of three glaucoma models.5 The mCherry CD3ζshRNA was injected into the vitreous body, then set up the glaucoma models,eyes were enucleated and processed for whole mount preparation. Brn3b-positive RGCs were counted and analyzed.Results1 We set up three types of glaucoma model:1)optic nerve crush model;2)NMDA intravitreal injection model;3)chornic high intraocular pressure model.2 RGC loss in optic nerve crush model focus on the postoperative week,approximately 97.4% above control(P<0.001).NMDA induced significant loss of RGC(approximately 94.7% above control) in 24 hours(P<0.001).High IOP triggered the death of 26.7%of RGC over a 4 week period(P<0.001).3 In Thyl-CFP mice with IOP elevation,three weeks after the IOP elevation,CFP-positive RGCs were reduced by 21%±4.8%(P<0.001).CFP-positive RGCs continued to decrease in number over time and 6 weeks after IOP elevation,CFP-positive RGCs were reduced by 30%±4.7%(P<0.001).In Thyl-YFP mice with IOP elevation,the damage on RGC dendrites start several days before cell death.4 We constructed the mCherry CD3ζshRNA expression vectors,CD3ζshRNA knockdown the CD3ζexpression level by 70% in HEK293 cells.5 CD3ζmRNA expression levels in retinal tissue of three glaucoma models are significantly changed compared to control(p<0.05),but CD3ζprotein expression levels in retinal tissue of three glaucoma models are not significantly changed compared to control.6 The damage of RGC in three glaucoma models with mCherry CD3ζshRNA introvitreal injection are not significantly decreased compared to the same models without mCherry CD3ζshRNA introvitreal injection.(p>0.05)Conclusions1 RGC loss in optic nerve crush model focus on the postoperative week, NMDA induced significant loss of RGC in 24 hours, high IOP trigger the progressive death of RGC over a long time.2 Confocal scanning laser microscopy is used to observe the RGC number decrease of Thy1-CFP mice and RGC dendrite loss of Thy1-YFP mice.3 CD3ζmRNA expression levels in retinal tissue of three glaucoma models are significantly changed compared to control,but CD3ζprotein expression levels in retinal tissue of three glaucoma models are not significantly changed compared to control. The damage of RGC in three glaucoma models with mCherry CD3ζshRNA introvitreai injection are not significantly decreased compared to the same models without mCherry CD3ζshRNA introvitreal injection.CD3ζmaybe not related to the RGC damage.更多还原