Effect Of Different Forms Of Light On Intracellular Concentration Of Ca²⁺ And Expression Of TGF-β₂ Of Guinea Pig Retinal Pigment Epithelial Cells In Vitro

Objective: To study the effect of different forms of light on Ca²⁺ and expression of transforming growth factor-β2(TGF-β2) of guinea pig retinal pigment epithelial (RPE) cells in vitro.Methods: Ten two-week-old healthy guinea pigs were chosen and RPE cells were cultured conventionally. Part of the cells were devided into verapamil (Ver) treated and untreated groups, then cells of each group were further divided into four groups: focused light group, defocused light group, parallel light group and control group. The first three groups were exposed to the focused light, defocused light (two forms of light were transformed from parallel light by passing through different lens) and the parallel light respectively, and control group was removed from exposure of light. Immediately after expoused to different forms of light, the fluorescence intensity of intracellular Ca²⁺ were detected by laser scanning confocal microscope(LSCM); Another part of cells were also divided in to focused light group, defocused light group, parallel light group and control group, the expression of TGF-β2 were observed by immunocytochemistry and the level of TGF-β2mRNA were detected by real time fluorescence quantitative PCR (RT-PCR) 24 hours after irradiation. Cool white light was used for light source. Cells were exposed to light with same spot diameter and degree of irradiation level (at 300 LUX) for 30 minutes respectively. Horizontal temperature of cells changed between 36.5℃³7.2℃. There was no natural light interference. In order to avoid the impaction of refraction of liquid, most of the medium were siphoned off before irradiation. The datas were analyzed by SPSS13.0 statistical software, statistical methods using completely randomized design ANOVA and Pearson linear correlation analysis. The forms-effect relations and the correlationship between the Ca²⁺ fluorescence intensity and the expression of TGF-β2 were explored.Results:1 Cells cultured and identification resultsRPE cells of guinea pigs began to adherence grow 24⁴8 hours after being isolated, grew actively after 5⁶ days and came to confluence after 7⁹ days. Passage cells began to adhere about 4 hours after being isolated and came to integration after 4⁶ days. Cells in culture were confirmed to be RPE cells by immunocytochemistry.2 Effect of Ver on apoptosis rate and the fluorescence intensity of Ca²⁺ of RPE cellsDifferent concentrations of Ver had a dose dependent effect on the inhibition of RPE cells. Treated with Ver for 12h, the apoptosis rates of RPE cells in 20mg/L, 40mg/L, 80mg/L concentration group had no significant difference compared with control group(P>0.05). Apoptosis of RPE cells in 120mg/L, 160mg/L concentration group was significant, which compared with control group was statistically significant (P<0.05). Ver could reduce the Ca²⁺ fluorescence intensity of RPE cell,and there was significantly statistical difference in 80 mg/L group (P<0.05).3 Effect of different forms of light on the Ca²⁺ fluorescence intensity of RPE cellThe Ca²⁺ fluorescence intensity of focused group of no Ver treated part was significantly higher than other groups, had significant differences compared with the other groups(P<0.05).After added 80mg/L Ver on the RPE cells for 12h, exposure to light did not significantly increase the Ca²⁺ fluorescence intensity, there was no significantly difference among the four groups (p>0.05).4 Expression of TGF-β2 of RPE cells (by immunocytochemistry)Expression of TGF-β2 were detected in each group, distributed in cytoplasm and nucleus, mainly in cytoplasm. With image analysis, the optical density (OD) values of each group from high to low were: focused light group: 0.38±0.01, defocused light group: 0.36±0.00, parallel light group: 0.34± 0.03, control group: 0.34±0.01. The OD values of focused light group were highest, followed by the defocused light group, the difference among focused light group and the other groups was significant (P<0.05), the same among defocused light group and the other groups. There was no statistical difference between parallel light group and control group (P>0.05).5 Expression of TGF-β2mRNA of RPE cell (by RT-PCR) There were expression of TGF-β2mRNA in each group. The relative quantifycation (RQ) value of expression of TGF-β2mRNA of RPE cells in each group from high to low were: focused light group: 4.58±0.19, defocused light group: 2.34±0.16, parallel light group: 1.78±0.09, control group: 0.92±0.05, there was significantly statistical difference among them (P<0.05).6 Correlation between expression of TGF-β2 and Ca²⁺ fluorescence intensity There were linear trend between Ca²⁺ fluorescence intensity and OD values ( by immunohistochemical), RQ values (by RT-PCR) of expression of TGF-β2, and pearson correlation coefficients were 0.808, 0.906 respectivly, which showed that correlations between them were statistically significant (P<0.05) .Conclusions:1 Focused light can significantly stimulate the concentration of intracellular Ca²⁺ and expression of TGF-β2 of RPE cells, defocused light also have the same impaction on the expression of TGF-β2 of RPE cells. There are significant correlations between Ca²⁺ fluorescence intensity and expression of TGF-β2 both in the protein and transcription level, which showed that Ca²⁺ may involved in the information transmission of TGF-β2 expression.2 Ver above a certain concentration can induce apoptosis of RPE cells, 80mg/L may not lead to apoptosis of RPE cells under the premise of effectively reduce intracellular Ca²⁺ fluorescence intensity.3 Different forms of light have no effect on the Ca²⁺ fluorescence intensity of RPE cells after treated with Ver(80mg/L) for 12h.