Effects of blue light on chemokines mRNA expressions in human retinal pigment epithelium cells at replicative senescence

1. Dr Song Chen
2. Dr Xiu-Bin Ma

Tianjin Eye Hospital

Objective: A blue light can induce human retinal pigment epithelium(RPE) cells to be retinal photo-damage. A study indicated that ultraviolet(UV) can make RPE cells replicative senescence. The damage of blue light is similar to UV. The aim of the experiment is to observe the effects of blue light on human RPE cells replicative senescence and expression of chemokines messenger ribonucleic acid(mRNA) in RPE cell.We want to find the relationship between replicative senescence and chemokines. It is to be discussed the role of blue light, RPE replicative senescence and inflammatory reaction in pathogenesis of age-related macular degeneration (AMD).

Methods:
1. After corneal transplantation, healthy human eyeball which came from human died by accident was used for experiments. Primary human RPE culture was harvested with trypsin digestion. Cell morphology and characterization were assessed by phase contrast microscopy. The cells were identified with immunohistochemistry.
2. The cells were distributed into experiment and control groups. The experiment groups were placed under exposure of blue light source of light emitting diode(LED). The control group was encapsulated by black paper during exposure.
3. All the groups from passage 3 to passage 5 were seeded at a density of 2¡Á105 cells/ml into 24 well culture plate for 24h to detect senescence associated beta-galactosidae(SA-¦Â-Gal) activity. RPE cells replicative senescence was defined of blue particles in plasma of cells. Positive cells were counted in 5 selected random high power microscope fields.
4. All the cells from passage 3 to passage 5 were seeded at a density of 1¡Á106 cells/ml into 6 well culture plates for 24h and followed by semiquantitative RT-PCR assay to detect expressions of chemokines mRNA in RPE cells.
5. All data was showed with mean¡Àstandard deviation ( ¡Às) and was dealt by SPSS 13.0. After test of normality and homogeneity test for variance, multiple factor analysis of variance was used if equal variance assumed followed by multiple comparisons with test of LSD-t. The relationship between replicative senescence and chemukines were dealt by Pearson correlation coefficient. The mean difference was significant at the level of 0.05(which is checked by professor zheng yuezhong who is from Tianjin eye hospital).

Results:
1. This primary culture resulted in cells was well-preserved morphology. some cells after adhere was round or irregular. It was rich in pigment granules. The next passages were grew well and were the pigment granules decreased obviousely. The result of immunohistochemistry was positive of the antigen of anti-keratin.
2. While the number of the replicative senescence cells with blue particles increased as the function of exposure intensity. The difference within the different light exposure intensity had statistics significance (P=0.0031). After two was dealt by LSD-t, we found that except for the difference between high light intensity and middle light intensity, every other two groups had statistics significance (P<0.05). Between different passages, the difference within the different passages had statistics significance (P=0.0094). Except for the difference between passage 4 and passage 5, the difference between every other two passage had statistics significance (P<0.05).
3. Different intensity of blue light was detected by semiquantitative RT-PCR assay. The exprressions chemokines mRNA in RPE cells increased as the increase of exposure intensity. The difference within the different light exposure intensity had statistics significance (P<0.01). After two was dealt by LSD-t, we found that except for the difference between high light intensity and middle light intensity, every other two groups had statistics significance (P<0.05). Between different passages, the difference within the different passages had statistics significance (P<0.01). Except for the difference between passage 4 and passage 5, the difference between every other two passage had statistics significance (P<0.05).
4. The chemokines in RPE cells were increased as the replicative senescence cells increased. The relation between replicative senescence and interleukin-8(IL-8) had statistics significance(r=0.736, P=0.0087). The relation between replicative senescence and monocyte chemoattractant protein-1(MCP-1) had statistics significance (r=0.682, P=0.015).

Conclusions:
1. Primary human RPE culture is harvested with trypsin digestion. They can become the source of the human RPE cell which are checked by immunohistochemistry. The cells grow well. They can be passaged normally. The morphology and number could meet the need of the experiments.
2. By SA-¦Â-Gal staining, we confirm the replicative senescence of human RPE cells cultured in vitro are positive correlation with blue light intensity and passage times and as the intensity of blue light and the passages increase, the replicative senescence increases.
3. The chomokins in human RPE cells was detected by semiquantitative RT-PCR assay. We find the blue light increases the expressions of chemokins in human RPE cells cultured in vitro. The expressions of chemokins increases with the increase of blue light intensity and passage times.
4. We confirm the expressions of chemokins increases with the increase of replicative senescence cells. The chemokins mRNA in the RPE cells which become replicative senescence increases.
5. These experiments firstly combine blue light, RPE cell replicative senescence, and chemokines together and provide held evidence for blue light induced RPE cells replicative senescence, blue light induced the chemokines in RPE cells, the chemokines mRNA in replicative senescence cells increases and explanation of the role of the exposure of blue light, RPE cell replicative senescence, inflammatory reaction in pathogenesis of AMD.
Keywords: blue light, retinal pigment epithelium (RPE), replicative senescence, IL-8, MCP-1