The pilot study on rapamycin retarding the differentiation of RPE cells in vitro

Abstract

Background Retinal pigment epithelial (RPE) cell transplantation is a novel approach to the treatment of hereditary retinal diseases, however, human-derived RPE cell line occurs de-differentiation during in vitro cell culture.Studies showed that early abnormal activation of Akt/mammalian target of rapamycin (mTOR) signal pathway is the primary cause of RPE cell line de-differentiation, therefore, the inhibition of mTOR pathway will be helpful for the retard of de-differentiation of RPE cells. Objective This study aimed to investigate whether rapamycin can suppress the activation of mTOR pathway and promote differentiation of ARPE19 cells. Methods ARPE-19 cells were incubated in 12-well plate and divided into control group and rapamycin-treated group.DMSO or rapamycin with the final concentration of 400 nmol/L was added in the medium of the control group and the rapamycin-treated group, respectively.The cells of each group were collected 24 hours and 48 hours after cultured.The expression of zonula occludens-1 (ZO-1) in the cells was examined by immunofluorescence.The relative expression levels of RPE cell specific genes and proteins were assayed by using real-time quantitative PCR and Western blot, respectively.The detected results were compared between the two groups. Results ZO-1 was expressed in both groups, but the fluorescence intensity was evidently enhanced in the rapamycin-treated group.The relative expression levels of RPE65, MERKT and LRAT mRNA in the cells increased by 25.97%, 29.71% and 13.00% in the rapamycin-treated group compared with the control group 24 hours after cultured (P=0.04, 0.04, 0.04), and the expression levels of RPE65, LRAT, rLBP1, BEST1, keratin18 and MERKT mRNA elevated by 174.00%, 88.00%, 56.18%, 193.81%, 10.83% and 35.02% in the rapamycin-treated group in comparison with the control group 48 hours after cultured (P=0.00, 0.04, 0.01, 0.04, 0.04, 0.03). In addition, the expressions of p-mTOR, p-P70S6 and p-S6 protein were weaker in the rapamycin-treated group than those in the control group both 24 hours and 48 hours after cultured.Twenty four hours after cultured, the expression level of ZO-1 protein raised by 40% in the rapamycin-treated group compared with the control group (P=0.01); while 48 hours after cultured, the expression levels of ZO-1, MERKT, catenin and LRAT proteins elevated by 36.00%, 57.37%, 13.68% and 41.07% in the rapamycin-treated group in comparison with the control group (P=0.01, 0.00, 0.04, 0.04). Conclusions Rapamycin can suppress the activation of mTOR signaling pathway and up-regulate the expressions of RPE specific genes in ARPE19 cells.Inhibition of mTOR pathway might be an effective way for culturing RPE cells in vitro. Key words: ARPE19 cells; Rapamycin; mTOR signaling pathway; Differentiation