小鼠慢性高眼压模型中CD3ζ基因对视网膜神经节细胞作用的研究

发布时间：2018-05-19 02:28

本文选题：青光眼 + 视网膜神经节细胞　；参考：《武汉大学》2013年博士论文

【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丢失的速度和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(intraocular 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,之前的研究己表明CD3是影响RGC树突形态和功能的关键因子,CD3小鼠的树突密度明显大于同龄野生型小鼠。CD3基因敲除小鼠RGC的细胞树突明显增加,CD3影响了生理状态下RGC树突的形态和功能,但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,观察缺血再灌注损伤情况下RGC的变化,研究CD3与RGC损伤的关系。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)烧灼小鼠角膜缘血管网和巩膜上静脉诱发的慢性高眼压模型；3)激光光凝角膜缘诱发的小鼠慢性高眼压模型。 2RGC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。 3通过共焦激光显微镜双通道激发观察缺血再灌注损伤情况下Thy1-CFP小鼠RGC变化的规律。 4Brn-3b阳性的RGC检测：建立青光眼模型,取出视网膜行全视网膜铺片,计数Brn-3b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)烧灼小鼠角膜缘血管网和巩膜上静脉诱导的慢性高眼压模型；3)激光光凝角膜缘诱发的慢性高眼压模型。 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96.8%(P0.001),烧灼小鼠角膜缘血管网和巩膜上静脉诱导的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25.0%(P0.001)。 3用Thy1-CFP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22%±4.8%(p0.001),6周后RGC减少30%±4.7%(p0.001)。 4缺血再灌注损伤导致Thy1-CFP小鼠RGC的持续死亡,CFP荧光蛋白是观察早期RGC变化的有效工具。 5CD3ζ基因敲除在小鼠慢性高眼压模型中对RGC的凋亡和减少没有明显影响和作用。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,烧灼小鼠角膜缘血管网和巩膜上静脉诱导的慢性高眼压模型诱导的RGC减少主要集中于术后24小时,激光光凝角膜缘诱发的慢性高眼压模型的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的数量减少失。 3缺血再灌注损伤模型是一种成熟的实验性神经变性动物模型,在本研究中通过观察再灌注后不同时间点CFP表达的RGCs的数量可以清晰地观察RGC凋亡和减少的趋势。通过观察Thy1-CFP自体荧光蛋白比起其他标记色方法可能在观察神经变性疾病中RGC的早期死亡方面更有优势,也更敏感。 4与野生型小鼠相比,CD3ζ基因敲除小鼠在慢性高眼压状态下的RGC的损伤没有明显减少,提示CD3ζ可能与RGC的损伤无关。
[Abstract]:Background of the study

In recent years , it has been shown that CD3 is the key factor affecting RGC ' s morphology and function . However , it has been shown that CD3 is the key factor affecting RGC ' s morphology and function .

Purpose

To establish an animal model of glaucoma , select the appropriate model living body to observe the change of RGC , observe the change of RGC under the condition of ischemia reperfusion injury , and study the relationship between CD3 and RGC injury .

method

The establishment of three glaucoma models : 1 ) optic nerve contusion model : the optic nerve is injured with forceps that can be automatically closed ;
2 ) The chronic high intraocular pressure model induced by vein and sclera of the cornea of the mouse was burned .
3 ) Mouse chronic high intraocular pressure model induced by laser photocoagulation .

2RGC injury observation : The quantitative changes of spontaneous fluorescence in Thy1 - cfp mice were observed in vivo by confocal laser microscopy .

3 The changes of RGC in Thy1 - cfp mice were observed by confocal laser microscope dual - channel excitation .

4Brn - 3b positive RGC detection : The glaucoma model was established , the whole retina was taken out of the retina , and the RGC positive for Brn - 3b was counted .

Results

1 ) Three kinds of glaucoma models were established : 1 ) the optic nerve injury model ;
2 ) The chronic high intraocular pressure model induced by vein and sclera of the cornea of the mouse was burned .
3 ) Chronic ocular hypertension model induced by laser photocoagulation .

RGC decreased by 96.8 % ( P0.001 ) at 7 days after operation , RGC decreased by 94.8 % ( P0.001 ) in 7 days , RGC decreased by 94.7 % ( P0.001 ) in 24 hours , RGC in chronic high intraocular pressure decreased continuously and RGC decreased by 25.0 % ( P0.001 ) in 4th week .

3 weeks after 3 weeks , the RGC decreased by 22 % 卤 4.8 % ( p 0.001 ) and 30 % 卤 4.7 % ( p 0.001 ) after 6 weeks .

4 Ischemia / reperfusion injury resulted in the continued death of the RGC in Thy1 - cfp mice , which was an effective tool for the observation of early RGC changes .

There was no significant influence and effect on the apoptosis and decrease of RGC in mouse chronic high intraocular pressure model .

Conclusion

The reduction of RGC induced by optic nerve contusion model was mainly focused on 24 hours after operation , and the RGC of chronic high intraocular pressure model induced by laser photocoagulation was mainly focused on 24 hours after operation . The reduction of RGC in chronic high intraocular pressure model induced by laser photocoagulation was sustained and slow , and the state of clinical glaucoma was more fit .

2 Reduction of the number of RGC in Thy1 - cfp mice that have established a chronic high intraocular pressure model was observed in vivo using confocal laser microscopy .

3 . The model of ischemia - reperfusion injury is a mature animal model of experimental neurodegeneration . In this study , the number of RGCs expressed at different time points after reperfusion can clearly observe the apoptosis and decrease of RGC . It is also more sensitive to observe the early death of RGC in neurodegenerative diseases compared with other labeling methods .

4 Compared with wild - type mice , the damage of RGC knockout mice in chronic high intraocular pressure was not significantly reduced , suggesting that CD3 zeta potential was not related to RGC injury .
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R-332;R774;R775

【参考文献】