577 Effects of UVA irradiation on the stemness and Opsin Expression of human amniotic mesenchymal stem cells in vitro

Abstract

Human amniotic mesenchymal stem cells amplified in vitro can be used for a variety of tissue and organ regeneration , and also has the potential to treat a variety of diseases. However MSCs cultured in vitro for a long time will weaken their self-renewal and multi differentiation ability . After long-term culture, MSCs became larger and flattened, and lost the ability to divide. Therefore, in vitro culture can reduce the pluripotency of MSCs. We chose UVA Irradiation to investigate the effect of OPN on the stemness of hAMSCs. Methods: hAMSCs were seeded onto cell Culture Dishes and irradiated with Ultraviolet A light at 0.25 J/cm2, 0.5 J/cm2, 5J/cm2, 10J/cm2, 20J/cm2 or not irradiated, The cell lysates were collected for further research 0, 12h, 24h , 48h, 72h after UVA irradiation. CCK-8 assay was used to measure cell viability, and apoptosis was evaluated by AnnexinV/PI, senescent cells were stained with a b-galactosidase staining kit. Expression of opsins and Sox2, OCT4, HIF-1α were evaluated by Western blotting.In this study, We found that 5J/cm2 irradiation with UVA induced no marked decrease of live cells and dead cells after 72H, Additionally, no remarkably increase of cell apoptosis and senescent cells were observed in UVA-irradiated cells as compared with non-irradiated control group with 5 J / cm2 . Western Blot to detect the expression of opsins showed that with the prolonged irradiation time, the expression of OPN3 did not increase significantly, The expression of OPN5 gradually increased with time, and the expression was significant at 72h, showing a time-dependent manner. Increased OCT4 and Sox2 that resulted from UVA irradiation at 72H were detected more than in control cultures. These results indicated that UVA irradiation improve OPN5 expression as well as hAMSCs stemness of OCT4 and Sox2.