本文選題：ARMS2蛋白 + 色素上皮��； 參考：《濟南大學》2011年碩士論文

【摘要】：目的 1、研究年齡相關性黃斑病變易感因子2 (age-related maculopathy susceptibility 2, ARMS2)蛋白在正常成人視網(wǎng)膜脈絡膜層表達位置以及在視網(wǎng)膜色素上皮細胞的分布。 2、從微觀水平研究H202誘導氧化應激對人視網(wǎng)膜色素上皮(ARPE-19)細胞內(nèi)ARMS2轉(zhuǎn)錄和蛋白水平的影響。初步探討ARMS2基因在氧化應激中對視網(wǎng)膜色素上皮細胞的作用。 方法 一、ARMS2蛋白在正常人眼組織中的表達 1、正常青壯年男性眼球10只,由青島眼科醫(yī)院眼庫提供。10只眼球均為死亡后立即摘除,離體后40C冷藏保存。瞳孔處于散大狀態(tài),用間接檢眼鏡觀察各眼球眼底,均無明顯異常。8小時內(nèi)在無菌狀態(tài)下,取下全角膜及角膜緣后4mm鞏膜,保留眼杯,40C冷藏保存。 2、取正常成人眼球3只,將其視網(wǎng)膜和脈絡膜組織制作冰凍切片。 3、采用免疫熒光和激光共聚焦顯微鏡技術,觀察ARMS2蛋白在視網(wǎng)膜脈絡膜各層的分布。 4、取正常成人眼球7只,進行視網(wǎng)膜色素上皮細胞原代培養(yǎng)。 5、應用免疫熒光顯微鏡檢測ARMS2蛋白在視網(wǎng)膜色素上皮細胞內(nèi)表達情況。 二、H202對人視網(wǎng)膜色素上皮細胞ARMS2表達影響 ]、培養(yǎng)ARPE-19細胞。 2、過氧化氫濃度0、100、300、500、700μmol/L作用ARPE-19細胞一定時間后,應用四甲基偶氮唑鹽比色法(MTT)檢測各濃度組H202作用2小時和4小時后ARPE-19細胞生長情況。 3、選擇H202作用ARPE-19細胞4小時作為觀察時間。采用實時定量Realtime PCR檢測ARMS2在轉(zhuǎn)錄水平變化,細胞免疫熒光法檢測蛋白水平變化。 4、MTT細胞活性結果采用單因素方差分析和最小顯著性差異法檢驗,過氧化氫濃度與細胞活性進行曲線估計。Realtime PCR和細胞免疫熒光法檢測ARMS2轉(zhuǎn)錄和蛋白水平變化均采用單因素方差分析和最小顯著性差異法檢驗。以P0.05作為差異有統(tǒng)計學意義。 結果 1、3只眼球均在視網(wǎng)膜血管、色素上皮層、Bruch膜、脈絡膜血管內(nèi)顯示有ARMS2陽性蛋白分布,而在神經(jīng)節(jié)細胞層、內(nèi)核層、外叢狀層、外核層、內(nèi)叢狀層陽性蛋白少； 2、7只眼球的視網(wǎng)膜色素上皮細胞原代培養(yǎng)均顯示,在視網(wǎng)膜色素上皮細胞內(nèi)ARMS2陽性蛋白呈簇狀,散在分布于細胞質(zhì)中。 1、MTT法檢測各組細胞生長狀況。H202濃度0μmol/L, 100μmol/L,300μmol/L, 500μmol/L,700μmol/L,結果用490nm吸光度值(A)平均值±標準差表示分別為： 2小時為0.302±0.036,0.298±0.005,0.328±0.013,0.292±0.287,0.318±0.021；4小時為0.531±0.037,0.370±0.017,0.371±0.016,0.33±0.006,0.297±0.012。2小時F=0.550,P=0.704；4小時F=6.782,P=0.007, P0.01，H2O2處理細胞2小時對細胞活性影響差異沒有統(tǒng)計學意義,而4小時對細胞活性影響差異有統(tǒng)計學意義,因此后續(xù)試驗均選擇H202作用ARPE-19細胞4小時。對H202濃度與細胞活性進行曲線估計,分析結果顯示r=-0.99,P0.05。說明在0-700μmol/L范圍內(nèi)隨H202濃度的升高ARPE-19細胞活性降低,且H202濃度與細胞活性呈高度負相關。2、Realtime PCR結果顯示不同濃度H202誘導ARMS2基因mRNA表達量分別為：1.154±0.007,1.324±0.022,1.350±0.011,1.280±0.031,升高分別為6.9%、23%、26%、19%,F=33.409,P=0.000,差異有顯著的統(tǒng)計學意義。在一定范圍內(nèi)隨H2O2濃度的升高而升高,在300-500μmol/L H2O2范圍內(nèi)ARMS2基因mRNA表達量達到最高值,之后隨濃度的上升而下降。 3、細胞免疫熒光檢測結果顯示H2O2誘導ARMS2蛋白表達量各組灰度值分別為：7320±493.428,14300±848.904,22400±1596.403,23400±2405.046,19200±561.373,單因素方差分析顯示F=22.843,P=0.000,差異有統(tǒng)計學意義。在一定范圍內(nèi)隨H2O2濃度的增加ARMS2蛋白表達量增加,在300-500μmol/L范圍內(nèi)ARMS2蛋白表達量達到最高峰,之后隨濃度的升高而降低。 結論 1、ARMS2蛋白在成年人正常眼視網(wǎng)膜血管、視網(wǎng)膜色素上皮層、Bruch膜、脈絡膜血管內(nèi)均有較高量的表達,其它部位表達量微弱；在視網(wǎng)膜色素上皮細胞內(nèi)呈簇狀分布在細胞質(zhì)中。 2、H2O2在一定濃度范圍內(nèi)時,可誘導氧化應激,使ARPE-19細胞內(nèi)ARMS2轉(zhuǎn)錄和蛋白水平表達量增加,如果H2O2的濃度超過ARPE-19細胞耐受范圍,ARMS2基因表達量下降。
[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.

【學位授予單位】：濟南大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R774.1

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