本文選題：青光眼 + 視網(wǎng)膜神經(jīng)節(jié)細(xì)胞��； 參考：《武漢大學(xué)》2013年博士論文

【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,引起周邊視野的缺損直至中心視力消失。視網(wǎng)膜神經(jīng)節(jié)細(xì)胞(retinal ganglion cell, RGC)的凋亡是青光眼的最主要特征之一,但是RGC損傷的具體機(jī)制至今不明。如何減少青光眼RGC的損害是世界性難題,觀察RGC丟失的速度和形態(tài)的變化對(duì)揭示青光眼的病程有重要的臨床指導(dǎo)意義。傳統(tǒng)的研究RGC損傷的方法無(wú)法活體動(dòng)態(tài)監(jiān)測(cè)RGC的變化,目前國(guó)際上共焦激光顯微鏡活體觀察轉(zhuǎn)基因小鼠帶自發(fā)熒光的RGC變化是一種新的趨勢(shì),但是主要集中在觀察RGC數(shù)量的變化,最近有研究表明活體觀察猴和小鼠RGC樹突的可能性,但是還沒(méi)有研究涉及到病理狀態(tài)下,對(duì)RGC形態(tài)變化的觀察。眼內(nèi)壓(intraocular pressure,IOP)升高是青光眼發(fā)病的主要原因之一,但不能解釋青光眼發(fā)病的所有特征。近年來(lái)免疫系統(tǒng)參與青光眼發(fā)病的相關(guān)研究越來(lái)越受關(guān)注,之前的研究己表明CD3是影響RGC樹突形態(tài)和功能的關(guān)鍵因子,CD3小鼠的樹突密度明顯大于同齡野生型小鼠。CD3基因敲除小鼠RGC的細(xì)胞樹突明顯增加,CD3影響了生理狀態(tài)下RGC樹突的形態(tài)和功能,但病理狀態(tài)下是否導(dǎo)致了RGC的損傷有待進(jìn)一步研究。 目的 建立青光眼動(dòng)物模型,選擇合適的模型活體觀察RGC變化,觀察缺血再灌注損傷情況下RGC的變化,研究CD3與RGC損傷的關(guān)系。 方法 1三種青光眼模型的建立：1)視神經(jīng)挫傷模型：用能自動(dòng)閉合的鑷子夾傷視神經(jīng)；2)燒灼小鼠角膜緣血管網(wǎng)和鞏膜上靜脈誘發(fā)的慢性高眼壓模型；3)激光光凝角膜緣誘發(fā)的小鼠慢性高眼壓模型。 2RGC損傷的觀察：用共焦激光顯微鏡活體動(dòng)態(tài)觀察Thy1-CFP小鼠帶自發(fā)熒光的RGC的數(shù)量變化。 3通過(guò)共焦激光顯微鏡雙通道激發(fā)觀察缺血再灌注損傷情況下Thy1-CFP小鼠RGC變化的規(guī)律。 4Brn-3b陽(yáng)性的RGC檢測(cè)：建立青光眼模型,取出視網(wǎng)膜行全視網(wǎng)膜鋪片,計(jì)數(shù)Brn-3b陽(yáng)性的RGC。 結(jié)果 1建立了三種青光眼模型：1)視神經(jīng)損傷模型；2)燒灼小鼠角膜緣血管網(wǎng)和鞏膜上靜脈誘導(dǎo)的慢性高眼壓模型；3)激光光凝角膜緣誘發(fā)的慢性高眼壓模型。 2視神經(jīng)挫傷模型RGC減少主要集中于術(shù)后一周,7天時(shí)RGC減少96.8%(P0.001),燒灼小鼠角膜緣血管網(wǎng)和鞏膜上靜脈誘導(dǎo)的RGC減少主要集中于術(shù)后一天,24小時(shí)RGC減少94.7%(P0.001),慢性高眼壓導(dǎo)致的RGC減少持續(xù)緩慢,第4周RGC減少25.0%(P0.001)。 3用Thy1-CFP小鼠建立慢性高眼壓模型,活體觀察結(jié)果顯示,Thy1-CFP小鼠3周后RGC減少22%±4.8%(p0.001),6周后RGC減少30%±4.7%(p0.001)。 4缺血再灌注損傷導(dǎo)致Thy1-CFP小鼠RGC的持續(xù)死亡,CFP熒光蛋白是觀察早期RGC變化的有效工具。 5CD3ζ基因敲除在小鼠慢性高眼壓模型中對(duì)RGC的凋亡和減少?zèng)]有明顯影響和作用。 結(jié)論 1視神經(jīng)挫傷模型引起的RGC減少主要集中在術(shù)后一周,燒灼小鼠角膜緣血管網(wǎng)和鞏膜上靜脈誘導(dǎo)的慢性高眼壓模型誘導(dǎo)的RGC減少主要集中于術(shù)后24小時(shí),激光光凝角膜緣誘發(fā)的慢性高眼壓模型的RGC減少是持續(xù)緩慢的,更加擬合臨床青光眼的狀態(tài)。 2可利用共焦激光顯微鏡活體觀察已建立慢性高眼壓模型的Thy1-CFP小鼠的RGC的數(shù)量減少失。 3缺血再灌注損傷模型是一種成熟的實(shí)驗(yàn)性神經(jīng)變性動(dòng)物模型,在本研究中通過(guò)觀察再灌注后不同時(shí)間點(diǎn)CFP表達(dá)的RGCs的數(shù)量可以清晰地觀察RGC凋亡和減少的趨勢(shì)。通過(guò)觀察Thy1-CFP自體熒光蛋白比起其他標(biāo)記色方法可能在觀察神經(jīng)變性疾病中RGC的早期死亡方面更有優(yōu)勢(shì),也更敏感。 4與野生型小鼠相比,CD3ζ基因敲除小鼠在慢性高眼壓狀態(tài)下的RGC的損傷沒(méi)有明顯減少,提示CD3ζ可能與RGC的損傷無(wú)關(guān)。
[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 % 鹵 4.8 % ( p 0.001 ) and 30 % 鹵 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 .
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R-332;R774;R775

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相關(guān)期刊論文 前3條

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本文編號(hào)：1908319