ARMS2基因在正常成人眼底组织和细胞的定位和表达

发布时间：2018-04-17 19:15

本文选题：ARMS2蛋白 + 色素上皮　；参考：《济南大学》2011年硕士论文

【摘要】：目的 1、研究年龄相关性黄斑病变易感因子2 (age-related maculopathy susceptibility 2, ARMS2)蛋白在正常成人视网膜脉络膜层表达位置以及在视网膜色素上皮细胞的分布。 2、从微观水平研究H202诱导氧化应激对人视网膜色素上皮(ARPE-19)细胞内ARMS2转录和蛋白水平的影响。初步探讨ARMS2基因在氧化应激中对视网膜色素上皮细胞的作用。方法一、ARMS2蛋白在正常人眼组织中的表达 1、正常青壮年男性眼球10只,由青岛眼科医院眼库提供。10只眼球均为死亡后立即摘除,离体后40C冷藏保存。瞳孔处于散大状态,用间接检眼镜观察各眼球眼底,均无明显异常。8小时内在无菌状态下,取下全角膜及角膜缘后4mm巩膜,保留眼杯,40C冷藏保存。 2、取正常成人眼球3只,将其视网膜和脉络膜组织制作冰冻切片。 3、采用免疫荧光和激光共聚焦显微镜技术,观察ARMS2蛋白在视网膜脉络膜各层的分布。 4、取正常成人眼球7只,进行视网膜色素上皮细胞原代培养。 5、应用免疫荧光显微镜检测ARMS2蛋白在视网膜色素上皮细胞内表达情况。二、H202对人视网膜色素上皮细胞ARMS2表达影响 ]、培养ARPE-19细胞。 2、过氧化氢浓度0、100、300、500、700μmol/L作用ARPE-19细胞一定时间后,应用四甲基偶氮唑盐比色法(MTT)检测各浓度组H202作用2小时和4小时后ARPE-19细胞生长情况。 3、选择H202作用ARPE-19细胞4小时作为观察时间。采用实时定量Realtime PCR检测ARMS2在转录水平变化,细胞免疫荧光法检测蛋白水平变化。 4、MTT细胞活性结果采用单因素方差分析和最小显著性差异法检验,过氧化氢浓度与细胞活性进行曲线估计。Realtime PCR和细胞免疫荧光法检测ARMS2转录和蛋白水平变化均采用单因素方差分析和最小显著性差异法检验。以P0.05作为差异有统计学意义。结果 1、3只眼球均在视网膜血管、色素上皮层、Bruch膜、脉络膜血管内显示有ARMS2阳性蛋白分布,而在神经节细胞层、内核层、外丛状层、外核层、内丛状层阳性蛋白少； 2、7只眼球的视网膜色素上皮细胞原代培养均显示,在视网膜色素上皮细胞内ARMS2阳性蛋白呈簇状,散在分布于细胞质中。 1、MTT法检测各组细胞生长状况。H202浓度0μmol/L, 100μmol/L,300μmol/L, 500μmol/L,700μmol/L,结果用490nm吸光度值(A)平均值±标准差表示分别为： 2小时为0.302±0.036,0.298±0.005,0.328±0.013,0.292±0.287,0.318±0.021；4小时为0.531±0.037,0.370±0.017,0.371±0.016,0.33±0.006,0.297±0.012。2小时F=0.550,P=0.704；4小时F=6.782,P=0.007, P0.01，H2O2处理细胞2小时对细胞活性影响差异没有统计学意义,而4小时对细胞活性影响差异有统计学意义,因此后续试验均选择H202作用ARPE-19细胞4小时。对H202浓度与细胞活性进行曲线估计,分析结果显示r=-0.99,P0.05。说明在0-700μmol/L范围内随H202浓度的升高ARPE-19细胞活性降低,且H202浓度与细胞活性呈高度负相关。2、Realtime PCR结果显示不同浓度H202诱导ARMS2基因mRNA表达量分别为：1.154±0.007,1.324±0.022,1.350±0.011,1.280±0.031,升高分别为6.9%、23%、26%、19%,F=33.409,P=0.000,差异有显著的统计学意义。在一定范围内随H2O2浓度的升高而升高,在300-500μmol/L H2O2范围内ARMS2基因mRNA表达量达到最高值,之后随浓度的上升而下降。 3、细胞免疫荧光检测结果显示H2O2诱导ARMS2蛋白表达量各组灰度值分别为：7320±493.428,14300±848.904,22400±1596.403,23400±2405.046,19200±561.373,单因素方差分析显示F=22.843,P=0.000,差异有统计学意义。在一定范围内随H2O2浓度的增加ARMS2蛋白表达量增加,在300-500μmol/L范围内ARMS2蛋白表达量达到最高峰,之后随浓度的升高而降低。结论 1、ARMS2蛋白在成年人正常眼视网膜血管、视网膜色素上皮层、Bruch膜、脉络膜血管内均有较高量的表达,其它部位表达量微弱；在视网膜色素上皮细胞内呈簇状分布在细胞质中。 2、H2O2在一定浓度范围内时,可诱导氧化应激,使ARPE-19细胞内ARMS2转录和蛋白水平表达量增加,如果H2O2的浓度超过ARPE-19细胞耐受范围,ARMS2基因表达量下降。
[Abstract]:objective
1, study of age-related maculopathy susceptibility 2 (age-related maculopathy 2 susceptibility, ARMS2) expression and distribution in retinal pigment epithelial cells in normal adult choroid retinal protein.
2, induced oxidative stress on human retinal pigment epithelium from the micro level of H202 (ARPE-19) on the level of ARMS2 mRNA and protein in cells. To investigate the role of retinal pigment epithelial cells ARMS2 gene in oxidative stress.
Method
A, the expression of ARMS2 protein in normal human tissues
1 normal young men, 10 eyes from the eye bank Qingdao eye hospital provides.10 eyes were removed immediately after death, from the preservation of 40C refrigerated body. In the pupil scattered state, observe the eye fundus by indirect ophthalmoscope, were not significantly abnormal.8 hours under aseptic conditions, remove the whole cornea 4mm edge of the sclera, retain the eye cup, 40C refrigerated.
2, 3 eyes of normal adults, the retinal and choroidal tissue frozen sections were made.
3, by immunofluorescence and confocal laser scanning microscopy, observe the distribution of ARMS2 protein in retina and choroid.
4, 7 eyes of normal adults, retinal pigment epithelial cells in primary culture.
5, to detect the expression of ARMS2 protein by immunofluorescence microscopy in retinal pigment epithelial cell.
Two. The effect of H202 on the expression of ARMS2 in human retinal pigment epithelial cells
], in cultured ARPE-19 cells.
2, the concentration of hydrogen peroxide ARPE-19 cells 0100300500700 mol/L after a certain period of time, using four methyl thiazolyl tetrazolium colorimetric assay (MTT) for 2 hours the concentration of group H202 detection function and the growth of ARPE-19 cells after 4 hours.
3, the selection of ARPE-19 cells of H202 for 4 hours as the observation time. Using real-time quantitative Realtime detection of PCR ARMS2 changes at the transcriptional level, to detect the changes of protein level by immunofluorescence.
4, the activity of MTT cells results by single factor analysis of variance and least significant difference test, hydrogen peroxide concentration and cell activity of PCR and.Realtime change curve to estimate cell immunofluorescence detection of ARMS2 mRNA and protein levels were analyzed by one-way ANOVA and least significant difference test. Using P0.05 as the difference was statistically significant.
Result
1,3 eye ball in retinal vessels, pigment epithelium, Bruch membrane, choroidal vessel showed positive ARMS2 protein distribution, and the kernel in the ganglion cell layer, layer, outer plexiform layer, outer nuclear layer, inner plexiform layer and less positive protein;
2,7 eye of retinal pigment epithelial cells of primary culture showed that the ARMS2 protein is clustered in the retinal pigment epithelial cells scattered in the cytoplasm.
1, MTT method to detect the cells growth of.H202 concentration of 0 mol/L, 100 mol/L, 300 mol/L, 500 mol/L, 700 mol/L, the absorbance values of 490nm (A) average standard deviation respectively:
2 hours is 0.302 + 0.036,0.298 + 0.005,0.328 + 0.013,0.292 + 0.287,0.318 + 0.021; 4 hours was 0.531 + 0.037,0.370 + 0.017,0.371 + 0.016,0.33 + 0.006,0.297 + 0.012.2 F=0.550 P=0.704 hours, 4 hours; F=6.782, P=0.007, P0.01, H2O2 cells were treated for 2 hours on cell activity influence the difference was not statistically significant, but there was statistically significant for 4 hours cell activity differences, so the follow-up tests were selected ARPE-19 cells of H202 for 4 hours. The curve to estimate the H202 concentration and cell activity analysis showed that r=-0.99, P0.05. decreased with the increase of H202 concentration of ARPE-19 cell activity in the 0-700 mol/L range, and the concentration of H202 and the cell activity was negatively correlated with.2, Realtime PCR the results showed that different concentrations of H202 induced ARMS2 gene expression of mRNA were: 1.154 + 0.007,1.324 + 0.022,1.350 + 0.011,1.280 + 0.031, l don't score 6. 9%, 23%, 26%, 19%, F=33.409, P=0.000, the difference was statistically significant. In a certain range with the concentration of H2O2 increases in the 300-500 mol/L range of H2O2 ARMS2 mRNA gene expression reached the highest value, then decreased with the increase of concentration.
3, cell immunofluorescence results showed that the expression level of each gray value of ARMS2 protein induced by H2O2: 7320 + 493.42814300 + 848.90422400 + 1596.40323400 + 2405.04619200 + 561.373, single factor variance analysis showed that F=22.843, P=0.000, the difference was statistically significant. With the increase of the concentration of H2O2 increased the expression of ARMS2 protein in a certain range, in 300-500 the range of mol/L ARMS2 protein expression reached the peak, then decreased with the concentration increasing.
conclusion
1, ARMS2 protein in adult normal retinal blood vessels, retinal pigment epithelium, Bruch membrane, expressed a higher amount of choroidal vessels in other parts, the expression of the weak; with a cluster distribution in the cytoplasm in retinal pigment epithelial cells.
In 2, H2O2 in a certain range of concentration, can induce oxidative stress, the expression of ARPE-19 intracellular levels of ARMS2 mRNA and protein increased, if the concentration of H2O2 exceeds ARPE-19 cell tolerance range, the decrease of ARMS2 gene expression.

【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R774.1

【参考文献】