核蛋白NSA2在小鼠脈絡(luò)膜新生血管動(dòng)物模型眼底組織的表達(dá)
發(fā)布時(shí)間：2019-02-25 11:48
【摘要】：目的:脈絡(luò)膜新生血管(choroidal neovascularization,CNV)對(duì)視網(wǎng)膜有很大的破壞性,使視力出現(xiàn)不可逆下降或喪失。CNV的發(fā)生機(jī)制很復(fù)雜,至今仍不能很好的進(jìn)行較為透徹的闡明,目前對(duì)CNV的治療也存在局限。進(jìn)一步深入研究CNV發(fā)病機(jī)制具有重要意義。NSA2是真核細(xì)胞中核仁核糖體大亞基前體的構(gòu)成蛋白,近年被證實(shí)參與癌細(xì)胞分裂增殖。CNV是血管內(nèi)皮細(xì)胞、RPE細(xì)胞等細(xì)胞病理性分裂增殖的結(jié)果,NSA2在CNV眼底組織中的表達(dá)情況以及與CNV發(fā)生過(guò)程中的細(xì)胞分裂增殖是否相關(guān)目前還未見(jiàn)相關(guān)研究報(bào)道。本實(shí)驗(yàn)旨在探明NSA2在CNV中的表達(dá)情況,推測(cè)在CNV中的可能作用機(jī)制及意義。 方法:健康雌性C56BL/6小鼠,選用532nM的Nd:YAG激光,設(shè)置不同參數(shù)光凝處理眼底,14天后,從心臟灌注FITC-D(sigma,2×106,5mg/ml,10%明膠),取出眼球,作脈絡(luò)膜鋪片,熒光顯微鏡下觀察證實(shí)CNV動(dòng)物模型造模的成功性,選擇合適的參數(shù),確定CNV動(dòng)物模型制作的穩(wěn)定性和成功性。造模成功后,將第二批小鼠造模,分別在3天、5天、7天、10天、14天處死小鼠,取出眼球,去掉眼前段,提取眼后段mRNA,檢測(cè)NSA2 mRNA表達(dá),觀察其表達(dá)規(guī)律。第三批小鼠右眼底光凝處理作為CNV實(shí)驗(yàn)組眼,左眼作正常對(duì)照組眼;在NSA2 mRNA表達(dá)高峰時(shí)間點(diǎn),處死動(dòng)物,去除眼前段后qPCR檢測(cè)NSA2 mRNA表達(dá);眼球冰凍切片,免疫熒光檢測(cè)NSA2蛋白表達(dá)。 結(jié)果: 1、CNV動(dòng)物模型制作: 心臟灌注FITC-D,脈絡(luò)膜鋪片,在熒光顯微鏡下觀察:激光參數(shù)設(shè)置為直徑50um、曝光時(shí)間0.1s、能量200mW、每只眼光凝6點(diǎn)時(shí),CNV形成率較高,達(dá)75%;模型較穩(wěn)定,便于統(tǒng)計(jì)分析。 2、NSA2 mRNA在眼后段的表達(dá): qPCR檢測(cè)顯示在正常對(duì)照組眼和CNV實(shí)驗(yàn)組眼的眼后段都存在NSA2 mRNA表達(dá),CNV造模后第5天,NSA2 mRNA表達(dá)明顯升高(P0.05)。 3、NSA2蛋白在眼后段的表達(dá): 免疫熒光檢測(cè)顯示正常對(duì)照組眼后段:NSA2蛋白主要表達(dá)在鞏膜組織及周圍的筋膜組織,RPE細(xì)胞層陽(yáng)性表達(dá),在視網(wǎng)膜其它細(xì)胞層未發(fā)現(xiàn)陽(yáng)性表達(dá)。CNV實(shí)驗(yàn)組眼后段:CNV組織附近鞏膜組織細(xì)胞NSA2蛋白陽(yáng)性率升高(P0.05);CNV組織附近的視網(wǎng)膜神經(jīng)節(jié)細(xì)胞NSA2蛋白呈陽(yáng)性表達(dá)。 結(jié)論: 1.利用Nd:YAG激光可以成功制造出C56BL/6小鼠CNV動(dòng)物模型,制造模型速度快,成功率較高,模型較穩(wěn)定。 2. NSA2 mRNA在眼后段組織中存在表達(dá),CNV造模后早期開(kāi)始表達(dá)上調(diào),提示NSA2參與CNV形成過(guò)程,在CNV形成過(guò)程中有特定的生物學(xué)功能作用。 3. NSA2蛋白在正常小鼠眼底組織中只存在少量表達(dá),CNV造模后早期,CNV組織NSA2蛋白表達(dá)陽(yáng)性,CNV周圍組織表達(dá)增強(qiáng),提示NSA2基因參與了CNV病理過(guò)程,其作用可能與CNV中的細(xì)胞分裂增殖以及增強(qiáng)病理?xiàng)l件下的視網(wǎng)膜神經(jīng)節(jié)細(xì)胞的存活相關(guān)。
[Abstract]:Objective: choroidal neovascularization (choroidal neovascularization,CNV) is very destructive to the retina, causing irreversible decrease or loss of visual acuity. The current treatment of CNV also has limitations. It is important to further study the pathogenesis of CNV. NSA2 is the constituent protein of nucleolar ribosomal large subunit precursor in eukaryotic cells, and has been proved to be involved in the division and proliferation of cancer cells in recent years. CNV is a vascular endothelial cell. The results of pathological division and proliferation of RPE cells, the expression of NSA2 in the fundus of CNV and whether it is related to the cell division and proliferation of CNV have not been reported. The purpose of this study was to investigate the expression of NSA2 in CNV and to speculate its possible mechanism and significance in CNV. Methods: healthy female C56BL/6 mice were treated with Nd:YAG laser of 532nM. After 14 days of treatment, FITC-D (sigma,2 脳 106 mg / ml 10% gelatin) was perfused from the heart, and the eyeball was taken out for choroidal preparation. The success of CNV animal model was confirmed by fluorescence microscope. The stability and success of CNV animal model were determined by choosing appropriate parameters. The second group of mice were killed at 3 days, 5 days, 7 days, 10 days and 14 days respectively. The eyeballs were taken out, the anterior segment was removed, and the mRNA, of the posterior segment of the eyes was extracted to detect the expression of NSA2 mRNA and observe the expression of NSA2 mRNA. The third batch of mice were treated with photocoagulation of right eye fundus as CNV experimental group, and left eye as normal control group. At the peak time of NSA2 mRNA expression, the animals were killed and NSA2 mRNA expression was detected by qPCR after removing the anterior segment. The expression of NSA2 protein was detected by immunofluorescence. Results: 1. The animal model of FITC-D, was made: the heart was perfused with FITC-D, choroid slice, and observed under fluorescence microscope: laser parameters were set to 50 umum in diameter, exposure time was 0.1 s, energy was 200 MW, and each eye was coagulated at 6 o'clock. The formation rate of CNV was high (75%). The model is stable and convenient for statistical analysis. 2the expression of NSA2 mRNA in the posterior segment of the eyes: the expression of NSA2 mRNA in the posterior segment of the eyes of the normal control group and the experimental group of CNV was detected by qPCR, and the expression of NSA2 mRNA increased significantly on the 5th day after CNV (P0.05). 3The expression of NSA2 protein in the posterior segment of the eyes: the expression of NSA2 protein was mainly in the scleral tissue and the surrounding fascia tissue, and the positive expression of RPE cell layer was found in the normal control group. No positive expression was found in other layers of retinal cells. In CNV experimental group, the positive rate of NSA2 protein was increased in the scleral tissue near CNV (P0.05). The expression of NSA2 protein in retinal ganglion cells near CNV was positive. Conclusion: 1. The CNV animal model of C56BL/6 mice can be successfully made by using Nd:YAG laser. The model is fast, the success rate is high, and the model is stable. 2. NSA2 mRNA was expressed in the posterior segment of the eye, and up-regulated in the early stage of CNV modeling, suggesting that NSA2 is involved in the formation of CNV and has a specific biological function in the formation of CNV. 3. There was only a small amount of expression of NSA2 protein in the fundus of normal mice. In the early stage of CNV model, the expression of NSA2 protein was positive in CNV tissue, and the expression of NSA2 protein was increased in surrounding tissues of CNV, suggesting that NSA2 gene was involved in the pathological process of CNV. Its role may be related to cell division and proliferation in CNV and enhancement of survival of retinal ganglion cells under pathological conditions.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R773.4
本文編號(hào)：2430131
[Abstract]:Objective: choroidal neovascularization (choroidal neovascularization,CNV) is very destructive to the retina, causing irreversible decrease or loss of visual acuity. The current treatment of CNV also has limitations. It is important to further study the pathogenesis of CNV. NSA2 is the constituent protein of nucleolar ribosomal large subunit precursor in eukaryotic cells, and has been proved to be involved in the division and proliferation of cancer cells in recent years. CNV is a vascular endothelial cell. The results of pathological division and proliferation of RPE cells, the expression of NSA2 in the fundus of CNV and whether it is related to the cell division and proliferation of CNV have not been reported. The purpose of this study was to investigate the expression of NSA2 in CNV and to speculate its possible mechanism and significance in CNV. Methods: healthy female C56BL/6 mice were treated with Nd:YAG laser of 532nM. After 14 days of treatment, FITC-D (sigma,2 脳 106 mg / ml 10% gelatin) was perfused from the heart, and the eyeball was taken out for choroidal preparation. The success of CNV animal model was confirmed by fluorescence microscope. The stability and success of CNV animal model were determined by choosing appropriate parameters. The second group of mice were killed at 3 days, 5 days, 7 days, 10 days and 14 days respectively. The eyeballs were taken out, the anterior segment was removed, and the mRNA, of the posterior segment of the eyes was extracted to detect the expression of NSA2 mRNA and observe the expression of NSA2 mRNA. The third batch of mice were treated with photocoagulation of right eye fundus as CNV experimental group, and left eye as normal control group. At the peak time of NSA2 mRNA expression, the animals were killed and NSA2 mRNA expression was detected by qPCR after removing the anterior segment. The expression of NSA2 protein was detected by immunofluorescence. Results: 1. The animal model of FITC-D, was made: the heart was perfused with FITC-D, choroid slice, and observed under fluorescence microscope: laser parameters were set to 50 umum in diameter, exposure time was 0.1 s, energy was 200 MW, and each eye was coagulated at 6 o'clock. The formation rate of CNV was high (75%). The model is stable and convenient for statistical analysis. 2the expression of NSA2 mRNA in the posterior segment of the eyes: the expression of NSA2 mRNA in the posterior segment of the eyes of the normal control group and the experimental group of CNV was detected by qPCR, and the expression of NSA2 mRNA increased significantly on the 5th day after CNV (P0.05). 3The expression of NSA2 protein in the posterior segment of the eyes: the expression of NSA2 protein was mainly in the scleral tissue and the surrounding fascia tissue, and the positive expression of RPE cell layer was found in the normal control group. No positive expression was found in other layers of retinal cells. In CNV experimental group, the positive rate of NSA2 protein was increased in the scleral tissue near CNV (P0.05). The expression of NSA2 protein in retinal ganglion cells near CNV was positive. Conclusion: 1. The CNV animal model of C56BL/6 mice can be successfully made by using Nd:YAG laser. The model is fast, the success rate is high, and the model is stable. 2. NSA2 mRNA was expressed in the posterior segment of the eye, and up-regulated in the early stage of CNV modeling, suggesting that NSA2 is involved in the formation of CNV and has a specific biological function in the formation of CNV. 3. There was only a small amount of expression of NSA2 protein in the fundus of normal mice. In the early stage of CNV model, the expression of NSA2 protein was positive in CNV tissue, and the expression of NSA2 protein was increased in surrounding tissues of CNV, suggesting that NSA2 gene was involved in the pathological process of CNV. Its role may be related to cell division and proliferation in CNV and enhancement of survival of retinal ganglion cells under pathological conditions.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R773.4
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