小鼠青光眼模型視網(wǎng)膜神經(jīng)節(jié)細(xì)胞損傷與CD3ζ關(guān)系的實(shí)驗(yàn)研究
發(fā)布時(shí)間：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、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損傷的方法無法活體動(dòng)態(tài)監(jiān)測RGC的變化,目前國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,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(introcular 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。近年來免疫系統(tǒng)參與青光眼發(fā)病的相關(guān)研究越來越受關(guān)注。我們之前的研究已表明CD3δ是影響RGC樹突形態(tài)和功能的關(guān)鍵因子,CD3δ-/-小鼠的樹突密度明顯大于同齡野生型小鼠。ShRNA沉默CD3δ在RGC的表達(dá)會(huì)導(dǎo)致該細(xì)胞樹突明顯增加,CD3ζ影響了生理狀態(tài)下RGC樹突的形態(tài)和功能,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,構(gòu)建mCherry CD3ζshRNA表達(dá)載體,研究CD3ζ與RGC損傷的關(guān)系,探討RNAi技術(shù)在視神經(jīng)保護(hù)中的運(yùn)用效果。 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) NMDA誘導(dǎo)的視網(wǎng)膜損傷模型：玻璃體腔內(nèi)注射NMDA (40nmol)導(dǎo)致RGC凋亡3)慢性高眼壓模型：前房內(nèi)注射微珠(10x 106 microbeads/ml)堵塞前房角導(dǎo)致眼壓升高。 2 RGC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)量變化和和Thy1-YFP小鼠帶自發(fā)熒光的RGC的形態(tài)變化。 3 mCherry CD3ζshRNA表達(dá)載體構(gòu)建：mCherry CD3ζshRNA表達(dá)載體是由編碼shRNA的引物序列插入BamHI和XhoI位點(diǎn)之間。 4視網(wǎng)膜組織中CD3ζ的mRNA及蛋白表達(dá)水平檢測：RT-PCR和Western blot法檢測視網(wǎng)膜組織中CD3ζ的mRNA及蛋白表達(dá)水平。 5 Brn3b陽性的RGC檢測：將mCherry CD3ζshRNA注射入小鼠玻璃體腔后,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)Brn3b陽性的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) NMDA誘導(dǎo)的視網(wǎng)膜損傷模型3)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97.4%(P＜0.001),NMDA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26.7%(P0.001)。 3用Thy1-CFP小鼠和Thy1-YFP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21%±4.8%(P0.001),6周后RGC減少30%±4.7%(P0.001), Thy1-YFP小鼠的RGC的樹突丟失早于胞體消失。 4成功構(gòu)建了mCherry-CD3ζshRNA的表達(dá)載體,CD3ζshRNA減少HEK293的70%的CD3ζ表達(dá)。 5三種青光眼模型視網(wǎng)膜組織中CD3ζ的mRNA表達(dá)上升(P0.05),CD3ζ蛋白質(zhì)表達(dá)水平與正常視網(wǎng)膜相比沒有明顯變化。 6玻璃體腔注射mCherry CD3ζshRNA的三種青光眼模型中RGC的丟失沒有明顯減少(P0.05)。 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,NMDA誘導(dǎo)的RGC減少主要集中于術(shù)后24小時(shí),而前房內(nèi)注射微珠堵塞房角誘發(fā)的高眼壓模型的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)量減少和Thy1-YFP小鼠的RGC的樹突丟失。 3視神經(jīng)挫傷模型、NMDA誘導(dǎo)的視網(wǎng)膜損傷模型、慢性高眼壓模型的視網(wǎng)膜組織中CD3ζ的mRNA水平表達(dá)上升,但CD3ζ的蛋白表達(dá)水平無明顯變化,玻璃體腔注射mCherry CD3ζshRNA的的三種青光眼模型,RGC的損傷沒有明顯減少,提示CD3ζ能與RGC的損傷無關(guān)。
[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
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R775
本文編號(hào)：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
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R775
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相關(guān)期刊論文 前6條
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