核蛋白NSA2在小鼠脉络膜新生血管动物模型眼底组织的表达
发布时间：2019-02-25 11:48
【摘要】：目的:脉络膜新生血管(choroidal neovascularization,CNV)对视网膜有很大的破坏性,使视力出现不可逆下降或丧失。CNV的发生机制很复杂,至今仍不能很好的进行较为透彻的阐明,目前对CNV的治疗也存在局限。进一步深入研究CNV发病机制具有重要意义。NSA2是真核细胞中核仁核糖体大亚基前体的构成蛋白,近年被证实参与癌细胞分裂增殖。CNV是血管内皮细胞、RPE细胞等细胞病理性分裂增殖的结果,NSA2在CNV眼底组织中的表达情况以及与CNV发生过程中的细胞分裂增殖是否相关目前还未见相关研究报道。本实验旨在探明NSA2在CNV中的表达情况,推测在CNV中的可能作用机制及意义。 方法:健康雌性C56BL/6小鼠,选用532nM的Nd:YAG激光,设置不同参数光凝处理眼底,14天后,从心脏灌注FITC-D(sigma,2×106,5mg/ml,10%明胶),取出眼球,作脉络膜铺片,荧光显微镜下观察证实CNV动物模型造模的成功性,选择合适的参数,确定CNV动物模型制作的稳定性和成功性。造模成功后,将第二批小鼠造模,分别在3天、5天、7天、10天、14天处死小鼠,取出眼球,去掉眼前段,提取眼后段mRNA,检测NSA2 mRNA表达,观察其表达规律。第三批小鼠右眼底光凝处理作为CNV实验组眼,左眼作正常对照组眼;在NSA2 mRNA表达高峰时间点,处死动物,去除眼前段后qPCR检测NSA2 mRNA表达;眼球冰冻切片,免疫荧光检测NSA2蛋白表达。 结果: 1、CNV动物模型制作: 心脏灌注FITC-D,脉络膜铺片,在荧光显微镜下观察:激光参数设置为直径50um、曝光时间0.1s、能量200mW、每只眼光凝6点时,CNV形成率较高,达75%;模型较稳定,便于统计分析。 2、NSA2 mRNA在眼后段的表达: qPCR检测显示在正常对照组眼和CNV实验组眼的眼后段都存在NSA2 mRNA表达,CNV造模后第5天,NSA2 mRNA表达明显升高(P0.05)。 3、NSA2蛋白在眼后段的表达: 免疫荧光检测显示正常对照组眼后段:NSA2蛋白主要表达在巩膜组织及周围的筋膜组织,RPE细胞层阳性表达,在视网膜其它细胞层未发现阳性表达。CNV实验组眼后段:CNV组织附近巩膜组织细胞NSA2蛋白阳性率升高(P0.05);CNV组织附近的视网膜神经节细胞NSA2蛋白呈阳性表达。 结论: 1.利用Nd:YAG激光可以成功制造出C56BL/6小鼠CNV动物模型,制造模型速度快,成功率较高,模型较稳定。 2. NSA2 mRNA在眼后段组织中存在表达,CNV造模后早期开始表达上调,提示NSA2参与CNV形成过程,在CNV形成过程中有特定的生物学功能作用。 3. NSA2蛋白在正常小鼠眼底组织中只存在少量表达,CNV造模后早期,CNV组织NSA2蛋白表达阳性,CNV周围组织表达增强,提示NSA2基因参与了CNV病理过程,其作用可能与CNV中的细胞分裂增殖以及增强病理条件下的视网膜神经节细胞的存活相关。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R773.4
本文编号：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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R773.4
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