ARMS2 Iocalization And Expression In Retina Tissue And Cells

Objective1. To investigate the expression position of the age-related maculopathy susceptibility2 (ARMS2) protein in the normal adult retinochoroidal layers and the distribution of the protein in the retinal pigment epithelial cells.3. To explore the influence of oxidative stress induced by hydrogen peroxide on the age-related maculopathy susceptibility 2 (ARMS2) in human retinal pigment epithelial (ARPE-19) cells in transcription and protein levels, and to investigate the effects of oxidative stress on ARMS2 gene in the retinal pigment epithelial cells.Methods1. Expression of ARMS2 protein in the retina choroid layer and the retinal pigment epithelial cells1)10 normal young male eye-balls came from Qingdao Eye Hospital Eye Bank.10 eyes were removed immediately after death, in vitro 4℃refrigerated. The pupils were dilated, observed by indirect ophthalmoscope.No obvious abnormalities. Steriled condition within 8 hours, remove the entire cornea and limbus,4mm sclera, the eye cup to retain,40℃refrigerated.2) Frozen sections were made from retinal and choroidal tissue in 3 normal adult eyes.3) Use immunofluorescence and confocal laser microscope to observe the ARMS2 protein in the retina choroid layers of distribution. 4) Use 7 normal adult eye to bring up the retinal pigment epithelial cells.5) Use immunofluorescence microscopy to discover ARMS2 protein in retinal pigment epithelial cell expression.2. Effects of hydrogen peroxide on ARMS2 in human retinal pigment epithelial cells1)Bring up ARPE-19 cells.2) Hydrogen peroxide in the concentrations of 0,100,300,500,700μmol/l was used for ARPE-19 cells in a certain time,and then MTT was used to detect the effects at 2 and 4 hours.3) Changes of ARMS2 in transcription and protein levels were detected by realtime PCR and immunofluorescence.4) One ANOVA and LSD was used for data analysis, and P<0.05 was considered significantly different.Results1. The ARMS2 protein expresses higher in the retinal vessels, pigment epithelium, Bruch membrane and choroidal vasculature than the other layers.2. Primary culture of the retinal pigment epithelial cells show that the distribution of the ARMS2 protein is clustered in the cytoplasm.3. With hydrogen peroxide in the 0-700μmol/l for 4 hours, the activity of ARPE-19 decreased.4.Realtime PCR results showed that hydrogen peroxide led ARMS2 gene mRNA expression to increase. The CT values:1.154±0.007,1.324±0.022,1.350±0.011, 1.280±0.031, increasing 6.9%,23%,26%,19%, F=33.409,P<0.01. When the concentration of hydrogen peroxide increased to 300-500μmol/l, ARMS2 gene mRNA expression reached the highest value, and then decreased with the increase of hydrogen peroxide.5. Immunofluorescence results showed that hydrogen peroxide led ARMS2 protein expression to increase.The gray values:7320±493.428,14300±848.904, 22400±1596.403,23400±2405.046,19200±561.373. F=22.843,P<=0.000.When the concentration of hydrogen peroxide increased to 300-500μmol/l, ARMS2 protein expression reached the highest value, and then decreased with increase of hydrogen peroxide. The results in transcription and protein levels were same.Conclusion1. The ARMS2 protein expresses higher in the retinal vessels, pigment epithelium, Bruch membrane and choroidal vasculature than the other layers. The distribution of the ARMS2 protein is clustered in the cytoplasm in the retinal pigment epithelial cells.2. ARMS2 in ARPE-19 cells can increase in transcription and protein levels when the concentration of hydrogen peroxide increases.The expression of ARMS2 depends on the tolerance of ARPE-19.